Manipulation of Cytokine Levels Using CD83 Gene Products

ABSTRACT

The invention provides methods for modulating cytokine levels, GM-CSF levels and the immune system using CD83 nucleic acids, CD83 polypeptides, anti-CD83 antibodies and factors that influence CD83 activity or expression. The invention also provides mice having a mutant CD83 gene and mice having a transgenic CD83 gene, which are useful for defining the role of CD83 in the immune system and for identifying compounds that can modulate CD83 and the immune system.

This application is related to U.S. Application Ser. No. 60/331,958filed Nov. 21, 2001.

FIELD OF THE INVENTION

The invention relates to an altered CD83 gene product, and methods ofmodulating cytokine levels by modulating the expression of mutant andwild type CD83 gene products produced in a mammal. The invention alsorelates to the regulation of T cell and dendritic cell activity andconditions and treatments related thereto.

BACKGROUND OF THE INVENTION

CD83 is a 45 kilodalton glycoprotein that is predominantly expressed onthe surface of dendritic cells and other cells of the immune system.Structural analysis of the predicted amino acid sequence of CD83indicates that it is a member of the immunoglobulin superfamily. See,Zhou et al., J. Immunol. 149:735 (1992)). U.S. Pat. No. 5,316,920 and WO95/29236 disclose further information about CD83. While such informationsuggests that CD83 plays a role in the immune system, that role isundefined, and the interrelationship of CD83 with cellular factorsremains unclear.

Moreover, treatment of many diseases could benefit from more effectivemethods for increasing or decreasing the immune response. Hence, furtherinformation about how to modulate the immune system by using factorssuch as CD83 are needed.

SUMMARY OF THE INVENTION

The invention provides a method of modulating cytokine levels bymodulating the activity or expression of the CD83 gene products.According to the invention, cytokine levels can be modulated in a mammalor in mammalian cells that are involved in the immune response, forexample, antigen presenting cells or T cells.

The invention therefore provides a method of modulating cytokineproduction in a mammal or in an immune cell by modulating the activityor expression of a CD83 polypeptide. According to the invention, theproduction of a cytokine such as interleukin-2, interleukin-4, orinterleukin-10 can be modulated by modulating the activity or expressionof a CD83 polypeptide. In some embodiments, an antibody is used that canmodulate the activity or expression of a CD83 polypeptide. For example,the antibody can be administered to the mammal or the immune cell can becontacted with the antibody. In some embodiments, the immune cells are Tcells or antigen presenting cells. In other embodiments, the immunecells are CD4+ T cells.

The invention also provides a method of modulating granulocytemacrophage colony stimulating factor production in a mammal or in animmune cell by modulating the activity or expression of CD83polypeptides. In some embodiments, an antibody is used that can modulatethe activity or expression of a CD83 polypeptide. For example, theantibody can be administered to the mammal or the immune cell can becontacted with the antibody. In some embodiments, the immune cells are Tcells or antigen presenting cells. In other embodiments, the immunecells are CD4+ T cells.

The invention also provides a method of modulating tumor necrosis factorproduction in a mammal or in a mammalian cell by modulating the activityor expression of CD83 polypeptides. In some embodiments, an antibody isused that can modulate the activity or expression of a CD83 polypeptide.For example, the antibody can be administered to the mammal or themammalian cell can be contacted with the antibody. In some embodiments,the immune cells are T cells or antigen presenting cells. In otherembodiments, the immune cells are CD4+ T cells.

The invention further provides a method of inhibiting proliferation of ahuman peripheral blood mononuclear cell by modulating the activity orexpression of CD83 polypeptides. In some embodiments, an antibody isused that can modulate the activity or expression of a CD83 polypeptide.For example, the antibody can be administered to the mammal or the humanperipheral blood mononuclear cell can be contacted with the antibody.

The invention also provides an antibody that can bind to a CD83polypeptide comprising SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO:9, whereinactivated CD4⁺ T-cells produce lower levels of interleukin-4 when theT-cells are contacted with the antibody. The invention further providesan antibody that can bind to a CD83 polypeptide comprising SEQ ID NO:4,SEQ ID NO:8 or SEQ ID NO:9, wherein CD4⁺ T-cells proliferation isdecreased when the T-cells are contacted with the antibody. Such anantibody can have an amino acid sequence that includes SEQ ID NO:11, SEQID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62 or SEQ IDNO:64. Nucleic acids encoding such an antibody can have, for example, asequence that includes SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63 or SEQ ID NO:65.

The invention also provides a method for decreasing the activity of aCD83 gene product, comprising contacting the CD83 gene product with anantibody that comprises SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62 or SEQ ID NO:64. The activity of aCD83 gene product can be decreased in a mammal or in a cell that isinvolved in an immune response, for example, a T cell.

The invention further provides a method for decreasing the translationof a CD83 gene product in a mammalian cell, comprising contacting themammalian cell with a nucleic acid complementary to a CD83 nucleic acidcomprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:10.

In another embodiment, the invention provides a method for decreasingthe translation of a CD83 gene product in a mammal, comprisingadministering to the mammal a nucleic acid complementary to a CD83nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ IDNO:10.

The invention further provides a method for decreasing proliferation ofCD4+ T-cells in a mammal comprising administering to the mammal anantibody that can bind to a CD83 gene product, wherein the CD83 geneproduct comprises SEQ ID NO:2 or SEQ ID NO:9. The antibody can have asequence comprising SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62 or SEQ ID NO:64.

The invention also provides a method for decreasing interleukin-2 levelsand increasing interleukin-4 levels in a mammal comprising administeringto the mammal an antibody that can bind to a CD83 gene product, whereinthe CD83 gene product comprises SEQ ID NO:2 or SEQ ID NO:9. The antibodycan have a sequence comprising SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52,SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57,SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62 or SEQ ID NO:64.

The invention further provides a method for decreasing interleukin-2levels and increasing interleukin-4 levels in a mammal comprisingadministering to the mammal a nucleic acid complementary to a CD83nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ IDNO:10. In some embodiments the interleukin-2 levels are decreased andthe interleukin-4 levels are increased to treat an autoimmune disease.In other embodiments, the interleukin-2 levels are decreased and theinterleukin-4 levels are increased to stimulate production ofTh2-associated cytokines in transplant recipients, for example, toprolong survival of transplanted tissues.

The invention also provides a method for increasing interleukin-10levels in a mammal comprising administering to the mammal an antibodythat can bind to a CD83 gene product, wherein the CD83 gene productcomprises SEQ ID NO:2 or SEQ ID NO:9. The antibody can have a sequencecomprising SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62 or SEQ ID NO:64.

The invention further provides a method for increasing interleukin-10levels in a mammal comprising administering to the mammal a nucleic acidcomplementary to a CD83 nucleic acid comprising SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, or SEQ ID NO:10. In some embodiments, theinterleukin-10 levels are increased to treat neoplastic disease. Inother embodiments, the interleukin-10 levels are increased to treat atumor.

The invention also provides a method for increasing interleukin-2 levelsin a mammal comprising administering to the mammal a functional CD83polypeptide that comprises SEQ ID NO:9.

The invention further provides a method for increasing interleukin-2levels in a mammal comprising: (a) transforming a T cell from the mammalwith a nucleic acid encoding a functional CD83 polypeptide operablylinked to a promoter functional in a mammalian cell, to generate atransformed T cell; (b) administering the transformed T cell to themammal to provide increased levels of interleukin-2. In someembodiments, the CD83 polypeptide has a sequence that comprises SEQ IDNO:9 or the nucleic acid has a sequence that comprises SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, or SEQ ID NO:10. Such methods for increasinginterleukin-2 levels can be used to treat an allergy or an infectiousdisease.

The invention also provides a method for increasing granulocytemacrophage colony stimulating factor levels in a mammal comprisingadministering to the mammal an antibody that can bind to a CD83 geneproduct, wherein the CD83 gene product comprises SEQ ID NO:2 or SEQ IDNO:9.

Such an antibody can have a sequence comprising SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62 or SEQ IDNO:64.

The invention further provides a method for increasing granulocytemacrophage colony stimulating factor levels in a mammal comprisingadministering to the mammal a nucleic acid complementary to a CD83nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ IDNO:10.

The invention also provides a method for increasing tumor necrosisfactor levels at a selected site in a mammal comprising administering tothe site a functional CD83 polypeptide. In another embodiment, theinvention provides a method for increasing tumor necrosis factor levelsin a selected mammalian cell comprising transforming the cell with anucleic acid encoding a functional CD83 polypeptide. The CD83polypeptide employed can, for example, have a sequence comprising SEQ IDNO:9.

Mammals and birds may be treated by the methods and compositionsdescribed and claimed herein. Such mammals and birds include humans,dogs, cats, and livestock, for example, horses, cattle, sheep, goats,chickens, turkeys and the like.

The invention further provides a mutant mouse that can serve as ananimal model of diminished T cell activation or altered cytokine levels.The mutant mouse has an altered CD83 gene that produces a larger geneproduct, having SEQ ID NO:4 or containing SEQ ID NO:8. Also provided aremethods of using the mutant mouse model to study the effects ofcytokines on the immune system, inflammation, the function andregulation of CD83, T cell and dendritic cell activity, the immuneresponse and conditions and treatments related thereto. Hence, theinvention further provides a mutant mouse whose somatic and germ cellscomprise a mutant CD83 gene encoding a polypeptide comprising SEQ IDNO:4 or SEQ ID NO:8, wherein expression of the mutant CD83 gene reducesCD4+ T cell activation. The mutant CD83 gene can, for example, compriseSEQ ID NO:3.

The invention further provides a method of identifying a compound thatcan modulate CD4+ T cell activation comprising administering a testcompound to a mouse having a mutant or wild type transgenic CD83 geneand observing whether CD4+ T cell activation is decreased or increased.The somatic and/or germ cells of the mutant mouse can comprise a mutantCD83 gene encoding a polypeptide comprising SEQ ID NO:4 or SEQ ID NO:8.Alternatively, the somatic and/or germ cells of the mouse can contain awild type CD83 gene, for example, SEQ ID NO:1 or SEQ ID NO:9.

The invention also provides a mutant CD83 gene encoding a polypeptidecomprising SEQ ID NO:4 or SEQ ID NO:8. The invention further provides amutant CD83 gene comprising nucleotide sequence SEQ ID NO:3.

DESCRIPTION OF THE FIGURES

FIG. 1 provides flow cytometry data for G3 animals. As shown, reducednumbers of CD4+ T cells are seen in two animals from Pedigree 9, mouse9.4.1 and mouse 9.4.9. All other animals analyzed on that day exhibitnormal numbers of CD4+ T cells.

FIG. 2 provides a graph of flow cytometry data for G3 animals. Eachdiamond symbol represents an individual animal. As shown, multipleanimals from the N2 generation exhibit a reduced percentage of CD4+ Tcells.

FIG. 3 provides the nucleotide sequence of wild type mouse CD83 (SEQ IDNO:1). The ATG start codon and the TGA stop codon are underlined.

FIG. 4A-B provides the nucleotide sequence of the mutant CD83 gene (SEQID NO:3) of the invention derived from the mutant LCD4.1 animal. The ATGstart codon, the mutation and the TGA stop codon are underlined.

FIG. 5 provides the amino acid sequence for wild type (top, SEQ ID NO:2)and mutant (bottom, SEQ ID NO:4) CD83 coding regions. The additionalC-terminal sequences arising because of the CD83 mutation areunderlined.

FIG. 6A illustrates that dendritic cells from wild type (♦, WT DC) andmutant (▪, mutant DC) mice are capable of the allogeneic activation ofCD4+ T cells. CD4+ T cells were stimulated with 10,000, 1000 or 100dendritic cells for 5 days and proliferation measured by incorporationof tritiated thymidine.

FIG. 6B illustrates that CD4+ T cells from mutant mice (▪, mutant CD4)fail to respond to allogeneic stimulation with BALBc dendritic cells,although wild type animals (♦, WT CD4+) respond normally. CD4+ T cellswere stimulated with 10,000, 1000 or 100 dendritic cells for 5 days andproliferation measured by incorporation of tritiated thymidine.

FIG. 7 provides a bar graph illustrating IL-2, IL-4, IL-5, TNFα, andIFNγ production from wild type CD4+ T cells (white bar) or CD83 mutantCD4+ T cells (dark bar) that had been stimulated with 1 μg/ml ofanti-CD3 antibodies and 0.2 μg/ml of anti-CD28 antibodies for 72 hours.As illustrated, IL-2 levels are lower, and IL-4 levels are higher in theCD83 mutant T cells.

FIG. 8 provides a bar graph illustrating IL-10 production from wild typeCD4+ T cells (white bar) or CD83 mutant CD4+ T cells (dark bar) that hadbeen stimulated with 0.1 μg/ml of anti-CD28 antibodies and 1 to 10 μg/mlof anti-CD3 antibodies for 72 hours. As illustrated, IL-10 levels arehigher in the CD83 mutant T cells.

FIG. 9 provides a bar graph illustrating GM-CSF production from wildtype CD4+ T cells (white bar) or CD83 mutant CD4+ T cells (dark bar)that had been stimulated with anti-CD3 and anti-CD28 antibodies. Asillustrated, GM-CSF production is higher in the CD83 mutant cells thanin wild type cells.

FIG. 10A provides a bar graph illustrating IL-4 mRNA levels from wildtype CD4+ T cells (white bar) or CD83 mutant CD4+ T cells (dark bar)that had been stimulated with anti-CD3 and anti-CD28 antibodies. Asillustrated, the IL-4 mRNA levels are higher in the CD83 mutant cells.

FIG. 10B provides a bar graph illustrating IL-10 mRNA levels from wildtype CD4+ T cells (white bar) or CD83 mutant CD4+ T cells (dark bar)that had been stimulated with anti-CD3 and anti-CD28 antibodies. Asillustrated, the IL-10 mRNA levels are higher in the CD83 mutant cells.

FIG. 11 provides a graph illustrating that various preparations ofanti-CD83 antibodies inhibit IL-4 production in anti-CD3 and anti-CD28antibody stimulated T cells. The amount of IL-4 produced by T cells inpg/ml is plotted versus the concentration of different anti-CD83antibody preparations, including the 20B08 (♦) anti-CD83 preparation,the 20D04 (▪) anti-CD83 preparation, the 14C12 (▴) anti-CD83 preparationand the 11G05 (X) anti-CD83 antibody preparation.

FIG. 12 provides a graph illustrating that various preparations ofanti-CD83 antibodies inhibit T cell proliferation. The graph plots theincorporation of radioactive thymidine in cpms, which was used as anindicator of the amount of T cell proliferation, versus theconcentration of the different anti-CD83 antibody preparations,including the 20D04 (♦) anti-CD83 preparation, the 11G05 (▪) anti-CD83antibody preparation, the 14C12 (▴) anti-CD83 preparation and the 6G05anti-CD83 preparation (X).

FIG. 13 provides a graph illustrating that transgenic mice thatover-express wild type CD83 have increased T cell proliferation. Thegraph plots the incorporation of radioactive thymidine in cpms, whichwas used as an indicator of the amount of T cell proliferation, versusthe concentration of OVA peptide. The transgenic mice utilized had aT-cell receptor specific for chicken ovalbumin (OVA) 323-339 peptidethat can activate T-cells. When mixed with either transgenic or wildtype dendritic cells in the presence of OVA peptide, transgenic CD4+ Tcells had increased T-cell proliferation. However, transgenic dendriticcells could not substantially increase wild type CD4+ T cellproliferation. Transgenic CD83 CD4+ T cells mixed with wild typedendritic cells (♦); transgenic CD83 CD4+ T cells mixed with transgenicdendritic cells (▪); wild type CD4+ T cells mixed with transgenicdendritic cells (▴); and wild type CD4+ T cells mixed with wild typedendritic cells (X).

FIG. 14 provides a schematic diagram of the structural elements includedin the mouse CD83 protein used for generating antibodies.

FIG. 15 provides a graph of ELISA data illustrating the titer obtainedfor different isolates of polyclonal anti-CD83 anti-sera. The first (♦),second (▪) and third (▴) isolates had similar titers, though the titerof the second isolate (▪) was somewhat higher.

FIG. 16 illustrates that proliferation of PHA-activated human PBMCs wasinhibited by antibodies raised against the external region of the mouseCD83 protein (♦). Pre-immune serum (▪) had little effect on theproliferation of human PBMCs.

FIG. 17A provides a sequence alignment of anti-CD83 heavy chain variableregions isolated by the invention. Sequences for isolates 20B08H (SEQ IDNO:52), 6G05H (SEQ ID NO:53), 20D04H (SEQ ID NO:54), 11G05 (SEQ IDNO:66) and 14C12 (SEQ ID NO:67) are provided. The CDR regions arehighlighted in bold.

FIG. 17B provides a sequence alignment of anti-CD83 light chain variableregions isolated by the invention. Sequences for isolates 20B08H (SEQ IDNO:55), 6G05H (SEQ ID NO:56), 20D04H (SEQ ID NO:57), 11G05 (SEQ IDNO:68) and 14C12 (SEQ ID NO:69) are provided. The CDR regions arehighlighted in bold.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for modulating the immune system by usingCD83 proteins, CD83 nucleic acids and factors that modulate CD83activity or expression.

According to the invention, loss or reduction of CD83 activity in vivoresults in altered cytokine levels, for example, lower interleukin-2levels, increased interleukin-4 levels, increased GM-CSF levels andincreased interleukin-10 levels. Loss or reduction of CD83 activity invivo can also result in decreased numbers of T cells.

Moreover, the invention also relates to increased CD83 activity in vivothat can result in altered cytokine levels, for example, higherinterleukin-2 levels, decreased interleukin-4 levels, decreased GM-CSFlevels and decreased interleukin-10 levels. Increased CD83 expression oractivity in vitro and in vivo can also result in increased activationand increased numbers of T cells.

The effects of CD83 on the immune system, on GM-CSF and on cytokinelevels were analyzed by using mutant and transgenic mice. The mutantmouse has an altered CD83 gene that expresses altered (defective) CD83gene product. The transgenic mouse overexpresses CD83 gene products.Accordingly, the invention provides mammals such as mice that have amutant or wild type CD83 gene. These mice are useful for identifying therole that CD83 plays in the immune response. These mutant and transgenicanimals are useful for identifying factors for manipulating cytokinelevels and T cell activation by testing whether those factors andcompositions can modulate, inhibit or replace the activity of CD83 invivo.

CD83

CD83 is a lymphocyte and dendritic cell activation antigen that isexpressed by activated lymphocytes and dendritic cells. CD83 is also asingle-chain cell-surface glycoprotein with a molecular weight of about45,000 that is believed to be a member of the Ig superfamily. Thestructure predicted from the CD83 amino acid sequence indicates thatCD83 is a membrane glycoprotein with a single extracellular Ig-likedomain, a transmembrane domain and cytoplasmic domain of approximatelyforty amino acids. The mature CD83 protein has about 186 amino acids andis composed of a single extracellular V type immunoglobulin (Ig)-likedomain, a transmembrane domain and a thirty nine amino acid cytoplasmicdomain. Northern blot analysis has revealed that CD83 is translated fromthree mRNA transcripts of about 1.7, 2.0 and 2.5 kb that are expressedby lymphoblastoid cell lines. It is likely that CD83 undergoes extensivepost-translational processing because CD83 is expressed as a singlechain molecule, but the determined molecular weight is twice thepredicted size of the core protein. See U.S. Pat. No. 5,766,570.

An example of a human CD83 gene product that can be used in theinvention is provided below (SEQ ID NO:9):

  1 MSRGLQLLLL SCAYSLAPAT PEVKVACSED VDLPCTAPWD  41 PQVPYTVSWVKLLEGGEERM ETPQEDHLRG QHYHQKGQNG  81 SFDAPNERPY SLKIRNTTSC NSGTYRCTLQDPDGQRNLSG 121 KVILRVTGCP AQRKEETFKK YRAEIVLLLA LVIFYLTLII 161FTCKFARLQS IFPDFSKAGM ERAFLPVTSP NKHLGLVTPH 201 KTELVSuch a CD83 gene product can be encoded by a number of different nucleicacids. One example of a human CD83 nucleic acid is provided below (SEQID NO:10).

   1 CCTGGCGCAG CCGCAGCAGC GACGGCAGCG AACTCGGCCG   41 GGCCCGGGCGCGCGGGGGCG GGACGCGCAC GCGGCGAGGG   81 CGGCGGGTGA GCCGGGGGCG GGGACGGGGGCGGGACGGGG  121 GCGAAGGGGG CGGGGACGGG GGCGCCCGCC GGCCTAACGG  161GATTAGGAGG GCGCGCCACC CGCTTCCGCT GCCCGCCGGG  201 GAATCCCCCG GGTGGCGCCCAGGGAAGTTC CCGAACGGGC  241 GGGCATAAAA GGGCAGCCGC GCCGGCGCCC CACAGCTCTG 281 CAGCTCGTGG CAGCGGCGCA GCGCTCCAGC CATGTCGCGC  321 GGCCTCCAGCTTCTGCTCCT GAGCTGCGCC TACAGCCTGG  361 CTCCCGCGAC GCCGGAGGTG AAGGTGGCTTGCTCCGAAGA  401 TGTGGACTTG CCCTGCACCG CCCCCTGGGA TCCGCAGGTT  441CCCTACACGG TCTCCTGGGT CAAGTTATTG GAGGGTGGTG  481 AAGAGAGGAT GGAGACACCCCAGGAAGACC ACCTCAGGGG  521 ACAGCACTAT CATCAGAAGG GGCAAAATGG TTCTTTCGAC 561 GCCCCCAATG AAAGGCCCTA TTCCCTGAAG ATCCGAAACA  601 CTACCAGCTGCAACTCGGGG ACATACAGGT GCACTCTGCA  641 GGACCCGGAT GGGCAGAGAA ACCTAAGTGGCAAGGTGATC  681 TTGAGAGTGA CAGGATGCCC TGCACAGCGT AAAGAAGAGA  721CTTTTAAGAA ATACAGAGCG GAGATTGTCC TGCTGCTGGC  761 TCTGGTTATT TTCTACTTAACACTCATCAT TTTCACTTGT  801 AAGTTTGCAC GGCTACAGAG TATCTTCCCA GATTTTTCTA 841 AAGCTGGCAT GGAACGAGCT TTTCTCCCAG TTACCTCCCC  881 AAATAAGCATTTAGGGCTAG TGACTCCTCA CAAGACAGAA  921 CTGGTATGAG CAGGATTTCT GCAGGTTCTTCTTCCTGAAG  961 CTGAGGCTCA GGGGTGTGCC TGTCTGTTAC ACTGGAGGAG 1001AGAAGAATGA GCCTACGCTG AAGATGGCAT CCTGTGAAGT 1041 CCTTCACCTC ACTGAAAACATCTGGAAGGG GATCCCACCC 1081 CATTTTCTGT GGGCAGGCCT CGAAAACCAT CACATGACCA1121 CATAGCATGA GGCCACTGCT GCTTCTCCAT GGCCACCTTT 1161 TCAGCGATGTATGCAGCTAT CTGGTCAACC TCCTGGACAT 1201 TTTTTCAGTC ATATAAAAGC TATGGTGAGATGCAGCTGGA 1241 AAAGGGTCTT GGGAAATATG AATGCCCCCA GCTGGCCCGT 1281GACAGACTCC TGAGGACAGC TGTCCTCTTC TGCATCTTGG 1321 GGACATCTCT TTGAATTTTCTGTGTTTTGC TGTACCAGCC 1361 CAGATGTTTT ACGTCTGGGA GAAATTGACA GATCAAGCTG1401 TGAGACAGTG GGAAATATTT AGCAAATAAT TTCCTGGTGT 1441 GAAGGTCCTGCTATTACTAA GGAGTAATCT GTGTACAAAG 1481 AAATAACAAG TCGATGAACT ATTCCCCAGCAGGGTCTTTT 1521 CATCTGGGAA AGACATCCAT AAAGAAGCAA TAAAGAAGAG 1561TGCCACATTT ATTTTTATAT CTATATGTAC TTGTCAAAGA 1601 AGGTTTGTGT TTTTCTGCTTTTGAAATCTG TATCTGTAGT 1641 GAGATAGCAT TGTGAACTGA CAGGCAGCCT GGACATAGAG1681 AGGGAGAAGA AGTCAGAGAG GGTGACAAGA TAGAGAGCTA 1721 TTTAATGGCCGGCTGGAAAT GCTGGGCTGA CGGTGCAGTC 1761 TGGGTGCTCG CCCACTTGTC CCACTATCTGGGTGCATGAT 1801 CTTGAGCAAG TTCCTTCTGG TGTCTGCTTT CTCCATTGTA 1841AACCACAAGG CTGTTGCATG GGCTAATGAA GATCATATAC 1881 GTGAAAATTA TTTGAAAACATATAAAGCAC TATACAGATT 1921 CGAAACTCCA TTGAGTCATT ATCCTTGCTA TGATGATGGT1961 GTTTTGGGGA TGAGAGGGTG CTATCCATTT CTCATGTTTT 2001 CCATTGTTTGAAACAAAGAA GGTTACCAAG AAGCCTTTCC 2041 TGTAGCCTTC TGTAGGAATT CTTTTGGGGAAGTGAGGAAG 2081 CCAGGTCCAC GGTCTGTTCT TGAAGCAGTA GCCTAACACA 2121CTCCAAGATA TGGACACACG GGAGCCGCTG GCAGAAGGGA 2161 CTTCACGAAG TGTTGCATGGATGTTTTAGC CATTGTTGGC 2201 TTTCCCTTAT CAAACTTGGG CCCTTCCCTT CTTGGTTTCC2241 AAAGGCATTT ATTGCTGAGT TATATGTTCA CTGTCCCCCT 2281 AATATTAGGGAGTAAAACGG ATACCAAGTT GATTTAGTGT 2321 TTTTACCTCT GTCTTGGCTT TCATGTTATTAAACGTATGC 2361 ATGTGAAGAA GGGTGTTTTT CTGTTTTATA TTCAACTCAT 2401AAGACTTTGG GATAGGAAAA ATGAGTAATG GTTACTAGGC 2441 TTAATACCTG GGTGATTACATAATCTGTAC AACGAACCCC 2481 CATGATGTAA GTTTACCTAT GTAACAAACC TGCACTTATA2521 CCCATGAACT TAAAATGAAA GTTAAAAATA AAAAACATAT 2561 ACAAATAAAA AAAA

A sequence of a wild type mouse CD83 gene that can be used in theinvention is provided herein as SEQ ID NO:1. SEQ ID NO:1 is providedbelow with the ATG start codon and the TGA stop codon identified byunderlining.

   1 GCGCTCCAGC CGC ATG TCGC AAGGCCTCCA GCTCCTGTTT   41 CTAGGCTGCGCCTGCAGCCT GGCACCCGCG ATGGCGATGC   81 GGGAGGTGAC GGTGGCTTGC TCCGAGACCGCCGACTTGCC  121 TTGCACAGCG CCCTGGGACC CGCAGCTCTC CTATGCAGTG  161TCCTGGGCCA AGGTCTCCGA GAGTGGCACT GAGAGTGTGG  201 AGCTCCCGGA GAGCAAGCAAAACAGCTCCT TCGAGGCCCC  241 CAGGAGAAGG GCCTATTCCC TGACGATCCA AAACACTACC 281 ATCTGCAGCT CGGGCACCTA CAGGTGTGCC CTGCAGGAGC  321 TCGGAGGGCAGCGCAACTTG AGCGGCACCG TGGTTCTGAA  361 GGTGACAGGA TGCCCCAAGG AAGCTACAGAGTCAACTTTC  401 AGGAAGTACA GGGCAGAAGC TGTGTTGCTC TTCTCTCTGG  441TTGTTTTCTA CCTGACACTC ATCATTTTCA CCTGCAAATT  481 TGCACGACTA CAAAGCATTTTCCCAGATAT TTCTAAACCT  521 GGTACGGAAC AAGCTTTTCT TCCAGTCACC TCCCCAAGCA 561 AACATTTGGG GCCAGTGACC CTTCCTAAGA CAGAAACGGT  601 A TGA GTAGGATCTCCACTGG TTTTTACAAA GCCAAGGGCA  641 CATCAGATCA GTGTGCCTGA ATGCCACCCGGACAAGAGAA  681 GAATGAGCTC CATCCTCAGA TGGCAACCTT TCTTTGAAGT  721CCTTCACCTG ACAGTGGGCT CCACACTACT CCCTGACACA  761 GGGTCTTGAG CACCATCATATGATCACGAA GCATGGAGTA  801 TCACCGCTTC TCTGTGGCTG TCAGCTTAAT GTTTCATGTG 841 GCTATCTGGT CAACCTCGTG AGTGCTTTTC AGTCATCTAC  881 AAGCTATGGTGAGATGCAGG TGAAGCAGGG TCATGGGAAA  921 TTTGAACACT CTGAGCTGGC CCTGTGACAGACTCCTGAGG  961 ACAGCTGTCC TCTCCTACAT CTGGGATACA TCTCTTTGAA 1001TTTGTCCTGT TTCGTTGCAC CAGCCCAGAT GTCTCACATC 1041 TGGCGGAAAT TGACAGGCCAAGCTGTGAGC CAGTGGGAAA 1081 TATTTAGCAA ATAATTTCCC AGTGCGAAGG TCCTGCTATT1121 AGTAAGGAGT ATTATGTGTA CATAGAAATG AGAGGTCAGT 1161 GAACTATTCCCCAGCAGGGC CTTTTCATCT GGAAAAGACA 1201 TCCACAAAAG CAGCAATACA GAGGGATGCCACATTTATTT 1241 TTTTAATCTT CATGTACTTG TCAAAGAAGA ATTTTTCATG 1281TTTTTTCAAA GAAGTGTGTT TCTTTCCTTT TTTAAAATAT 1321 GAAGGTCTAG TTACATAGCATTGCTAGCTG ACAAGCAGCC 1361 TGAGAGAAGA TGGAGAATGT TCCTCAAAAT AGGGACAGCA1401 AGCTAGAAGC ACTGTACAGT GCCCTGCTGG GAAGGGCAGA 1441 CAATGGACTGAGAAACCAGA AGTCTGGCCA CAAGATTGTC 1481 TGTATGATTC TGGACGAGTC ACTTGTGGTTTTCACTCTCT 1521 GGTTAGTAAA CCAGATAGTT TAGTCTGGGT TGAATACAAT 1561GGATGTGAAG TTGCTTGGGG AAAGCTGAAT GTAGTGAATA 1601 CATTGGCAAC TCTACTGGGCTGTTACCTTG TTGATATCCT 1641 AGAGTTCTGG AGCTGAGCGA ATGCCTGTCA TATCTCAGCT1681 TGCCCATCAA TCCAAACACA GGAGGCTACA AAAAGGACAT 1721 GAGCATGGTCTTCTGTGTGA ACTCCTCCTG AGAAACGTGG 1761 AGACTGGCTC AGCGCTTTGC GCTTGAAGGACTAATCACAA 1801 GTTCTTGAAG ATATGGACCT AGGGGAGCTA TTGCGCCACG 1841ACAGGAGGAA GTTCTCAGAT GTTGCATTGA TGTAACATTG 1881 TTGCATTTCT TTAATGAGCTGGGCTCCTTC CTCATTTGCT 1921 TCCCAAAGAG ATTTTGTCCC ACTAATGGTG TGCCCATCAC1961 CCACACTATG AAAGTAAAAG GGATGCTGAG CAGATACAGC 2001 GTGCTTACCTCTCAGCCATG ACTTTCATGC TATTAAAAGA 2041 ATGCATGTGA A

Nucleic acids having SEQ ID NO:1 encode a mouse polypeptide having SEQID NO:2, provided below.

  1 MSQGLQLLFL GCACSLAPAM AMREVTVACS ETADLPCTAP  41 WDPQLSYAVSWAKVSESGTE SVELPESKQN SSFEAPRRRA  81 YSLTIQNTTI CSSGTYRCAL QELGGQRNLSGTVVLKVTGC 121 PKEAGESTFR KYRAEAVLLF SLVVFYLTLI IFTCKFARLQ 161SIFPDISKPG TEQAFLPVTS PSKHLGPVTL PKTETV

According to the invention, loss or reduction of CD83 activity in vivoresults in altered cytokine levels, for example, lower interleukin-2levels, increased interleukin-4 levels, increased GM-CSF levels andincreased interleukin-10 levels. Loss or reduction of CD83 activity invivo can also result in decreased numbers of T cells.

Moreover, increased CD83 activity in vivo can also result in alteredcytokine levels, for example, higher interleukin-2 levels, decreasedinterleukin-4 levels, decreased GM-CSF levels and decreasedinterleukin-10 levels. Increased CD83 expression or activity in vivo canalso result in increased activation or increased numbers of T cells.

The effect of CD83 on cytokine levels was ascertained through use of amutant mouse that encodes a mutant CD83. Such a mutant mouse has a CD83gene encoding SEQ ID NO:4, with added C-terminal sequences provided bySEQ ID NO:8. In contrast to these wild type CD83 nucleic acids andpolypeptides, the mutant CD83 gene of the invention has SEQ ID NO:3. SEQID NO:3 is provided below with the ATG start codon, the mutation, andthe TGA stop codon are identified by underlining.

   1 GCGCTCCAGC CGC ATG TCGC AAGGCCTCCA GCTCCTGTTT   41 CTAGGCTGCGCCTGCAGCCT GGCACCCGCG ATGGCGATGC   81 GGGAGGTGAC GGTGGCTTGC TCCGAGACCGCCGACTTGCC  121 TTGCACAGCG CCCTGGGACC CGCAGCTCTC CTATGCAGTG  161TCCTGGGCCA AGGTCTCCGA GAGTGGCACT GAGAGTGTGG  201 AGCTCCCGGA GAGCAAGCAAAACAGCTCCT TCGAGGCCCC  241 CAGGAGAAGG GCCTATTCCC TGACGATCCA AAACACTACC 281 ATCTGCAGCT CGGGCACCTA CAGGTGTGCC CTGCAGGAGC  321 TCGGAGGGCAGCGCAACTTG AGCGGCACCG TGGTTCTGAA  361 GGTGACAGGA TGCCCCAAGG AAGCTACAGAGTCAACTTTC  401 AGGAAGTACA GGGCAGAAGC TGTGTTGCTC TTCTCTCTGG  441TTGTTTTCTA CCTGACACTC ATCATTTTCA CCTGCAAATT  481 TGCACGACTA CAAAGCATTTTCCCAGATAT TTCTAAACCT  521 GGTACGGAAC AAGCTTTTCT TCCAGTCACC TCCCCAAGCA 561 AACATTTGGG GCCAGTGACC CTTCCTAAGA CAGAAACGGT  601 A A GAGTAGGATCTCCACTGG TTTTTACAAA GCCAAGGGCA  641 CATCAGATCA GTGTGCCTGA ATGCCACCCGGACAAGAGAA  681 GAATGAGCTC CATCCTCAGA TGGCAACCTT TCTTTGAAGT  721CCTTCACCTG ACAGTGGGCT CCACACTACT CCCTGACACA  761 GGGTCT TGA G CACCATCATATGATCACGAA GCATGGAGTA  801 TCACCGCTTC TCTGTGGCTG TCAGCTTAAT GTTTCATGTG 841 GCTATCTGGT CAACCTCGTG AGTGCTTTTC AGTCATCTAC  881 AAGCTATGGTGAGATGCAGG TGAAGCAGGG TCATGGGAAA  921 TTTGAACACT CTGAGCTGGC CCTGTGACAGACTCCTGAGG  961 ACAGCTGTCC TCTCCTACAT CTGGGATACA TCTCTTTGAA 1001TTTGTCCTGT TTCGTTGCAC CAGCCCAGAT GTCTCACATC 1041 TGGCGGAAAT TGACAGGCCAAGCTGTGAGC CAGTGGGAAA 1081 TATTTAGCAA ATAATTTCCC AGTGCGAAGG TCCTGCTATT1121 AGTAAGGAGT ATTATGTGTA CATAGAAATG AGAGGTCAGT 1161 GAACTATTCCCCAGCAGGGC CTTTTCATCT GGAAAAGACA 1201 TCCACAAAAG CAGCAATACA GAGGGATGCCACATTTATTT 1241 TTTTAATCTT CATGTACTTG TCAAAGAAGA ATTTTTCATG 1281TTTTTTCAAA GAAGTGTGTT TCTTTCCTTT TTTAAAATAT 1321 GAAGGTCTAG TTACATAGCATTGCTAGCTG ACAAGCAGCC 1361 TGAGAGAAGA TGGAGAATGT TCCTCAAAAT AGGGACAGCA1401 AGCTAGAAGC ACTGTACAGT GCCCTGCTGG GAAGGGCAGA 1441 CAATGGACTGAGAAACCAGA AGTCTGGCCA CAAGATTGTC 1481 TGTATGATTC TGGACGAGTC ACTTGTGGTTTTCACTCTCT 1521 GGTTAGTAAA CCAGATAGTT TAGTCTGGGT TGAATACAAT 1561GGATGTGAAG TTGCTTGGGG AAAGCTGAAT GTAGTGAATA 1601 CATTGGCAAC TCTACTGGGCTGTTACCTTG TTGATATCCT 1641 AGAGTTCTGG AGCTGAGCGA ATGCCTGTCA TATCTCAGCT1681 TGCCCATCAA TCCAAACACA GGAGGCTACA AAAAGGACAT 1721 GAGCATGGTCTTCTGTGTGA ACTCCTCCTG AGAAACGTGG 1761 AGACTGGCTC AGCGCTTTGC GCTTGAAGGACTAATCACAA 1801 GTTCTTGAAG ATATGGACCT AGGGGAGCTA TTGCGCCACG 1841ACAGGAGGAA GTTCTCAGAT GTTGCATTGA TGTAACATTG 1881 TTGCATTTCT TTAATGAGCTGGGCTCCTTC CTCATTTGCT 1921 TCCCAAAGAG ATTTTGTCCC ACTAATGGTG TGCCCATCAC1961 CCACACTATG AAAGTAAAAG GGATGCTGAG CAGATACAGC 2001 GTGCTTACCTCTCAGCCATG ACTTTCATGC TATTAAAAGA 2041 ATGCATGTGA AThe change from a thymidine in SEQ ID NO:1 to an adenine in SEQ ID NO:3at the indicated position (602) leads to read-through translationbecause the stop codon at positions 602-604 in SEQ ID NO:1 is changed toa codon that encodes an arginine. Accordingly, mutant CD83 nucleic acidshaving SEQ ID NO:3 encode an elongated polypeptide having SEQ ID NO:4,provided below, where the extra amino acids are underlined.

  1 MSQGLQLLFL GCACSLAPAM AMREVTVACS ETADLPCTAP  41 WDPQLSYAVSWAKVSESGTE SVELPESKQN SSFEAPRRRA  81 YSLTIQNTTI CSSGTYRCAL QELGGQRNLSGTVVLKVTGC 121 PKEATESTFR KYRAEAVLLF SLVVFYLTLI IFTCKFARLQ 161SIFPDISKPG TEQAFLPVTS PSKHLGPVTL PKTETVRVGS 201PLVFTKPRAH QISVPECHPD KRRMSSILRW QPFFEVLHLT 241 VGSTLLPDTG S

In another embodiment, the invention provides mutant CD83 nucleic acidsthat include SEQ ID NO:5.

  1 ATG TCGCAAG GCCTCCAGCT CCTGTTTCTA GGCTGCGCCT  41 GCAGCCTGGCACCCGCGATG GCGATGCGGG AGGTGACGGT  81 GGCTTGCTCC GAGACCGCCG ACTTGCCTTGCACAGCGCCC 121 TGGGACCCGC AGCTCTCCTA TGCAGTGTCC TGGGCCAAGG 161TCTCCGAGAG TGGCACTGAG AGTGTGGAGC TCCCGGAGAG 201 CAAGCAAAAC AGCTCCTTCGAGGCCCCCAG GAGAAGGGCC 241 TATTCCCTGA CGATCCAAAA CACTACCATC TGCAGCTCGG281 GCACCTACAG GTGTGCCCTG CAGGAGCTCG GAGGGCAGCG 321 CAACTTGAGCGGCACCGTGG TTCTGAAGGT GACAGGATGC 361 CCCAAGGAAG CTACAGAGTC AACTTTCAGGAAGTACAGGG 401 CAGAAGCTGT GTTGCTCTTC TCTCTGGTTG TTTTCTACCT 441GACACTCATC ATTTTCACCT GCAAATTTGC ACGACTACAA 481 AGCATTTTCC CAGATATTTCTAAACCTGGT ACGGAACAAG 521 CTTTTCTTCC AGTCACCTCC CCAAGCAAAC ATTTGGGGCC561 AGTGACCCTT CCTAAGACAG AAACGGTAAG AGTAGGATCT 601 CCACTGGTTTTTACAAAGCC AAGGGCACAT CAGATCAGTG 641 TGCCTGAATG CCACCCGGAC AAGAGAAGAATGAGCTCCAT 681 CCTCAGATGG CAACCTTTCT TTGAAGTCCT TCACCTGACA 721GTGGGCTCCA CACTACTCCC TGACACAGGG TCT TGANucleic acids having SEQ ID NO:5 also encode a polypeptide having SEQ IDNO:4.

In another embodiment, the invention provides mutant CD83 nucleic acidsthat include SEQ ID NO:7.

  1 A GAGTAGGAT CTCCACTGGT TTTTACAAAG CCAAGGGCAC  41 ATCAGATCAGTGTGCCTGAA TGCCACCCGG ACAAGAGAAG  81 AATGAGCTCC ATCCTCAGAT GGCAACCTTTCTTTGAAGTC 121 CTTCACCTGA CAGTGGGCTC CACACTACTC CCTGACACAG 161 GGTCT TGA

The invention also provides a mutant CD83 containing SEQ ID NO:8,provided below.

1 RVGSPLVFTK PRAHQISVPE CHPDKRRMSS ILRWQPFFEV

41 LHLTVGSTLL PDTGS

SEQ ID NO:8 contains read through sequences that are not present in thewild type CD83 polypeptide but are present in the mutant CD83 geneproduct provided by the invention.

CD83 Modulation of Cytokine Levels

The invention also provides compositions and methods for increasinginterleukin-4 levels, increasing GM-CSF levels, increasinginterleukin-10 levels and decreasing interleukin-2 levels in a mammal.Such compositions and methods generally operate by decreasing theexpression or function of CD83 gene products in the mammal.Interleukin-4 promotes the differentiation of Th2 cells while decreasingthe differentiation of precursor cells into Th1 cells. Th2 cells areinvolved in helping B lymphocytes and in stimulating production of IgG1and IgE antibodies. Enhancement of Th2 formation may be useful, forexample, in autoimmune diseases and in organ transplantation.

Alternatively, the invention provides compositions and methods fordecreasing interleukin-4 levels, decreasing interleukin-10 levels andincreasing interleukin-2 levels in a mammal. Such compositions andmethods generally increase the expression or function of CD83 geneproducts in the mammal. Interleukin-2 promotes the differentiation ofTh1 cells and decreases the differentiation of Th-2 cells. Th1 cellsare, for example, involved in inducing autoimmune and delayed typehypersensitivity responses. Inhibition of Th2 formation may be useful intreating allergic diseases, malignancies and infectious diseases.

CD4+ T helper cells are not a homogeneous population but can be dividedon the basis of cytokine secretion into at least two subsets termed Thelper type 1 (Th1) and T helper type 2 (Th2) (see e.g., Mosmann, T. R.et al. (1986) J. Immunol. 136:2348-2357; Paul, W. E. and Seder, R. A.(1994) Cell 76:241-251; Seder, R. A. and Paul, W. E. (1994) Ann. Rev.Immunol. 12:635-673). Th1 cells secrete interleukin-2 (IL-2) andinterferon-γ (IFN-γ) while Th2 cells produce interleukin-4 (IL4),interleukin-5 (IL-5), interleukin-10 (IL-10) and interleukin-13 (IL-13).Both subsets produce cytokines such as tumor necrosis factor (TNF) andgranulocyte/macrophage-colony stimulating factor (GM-CSF).

In addition to their different pattern of cytokine expression, Th1 andTh2 cells are thought to have differing functional activities. Forexample, Th1 cells are involved in inducing delayed typehypersensitivity responses, whereas Th2 cells are involved in providingefficient “help” to B lymphocytes and stimulating production of IgG1 andIgE antibodies.

The ratio of Th1 to Th2 cells is highly relevant to the outcome of awide array of immunologically-mediated clinical diseases includingautoimmune, allergic and infectious diseases. For example, inexperimental leishmania infections in mice, animals that are resistantto infection mount predominantly a Th1 response, whereas animals thatare susceptible to progressive infection mount predominantly a Th2response (Heinzel, F. P., et al. (1989) J. Exp. Med. 169:59-72;Locksley, R. M. and Scott, P. (1992) Immunoparasitology Today 1:A58-A61). In murine schistosomiasis, a Th1 to Th2 switch is observedcoincident with the release of eggs into the tissues by female parasitesand is associated with a worsening of the disease condition (Pearce, E.J., et al. (1991) J. Exp. Med. 173:159-166; Grzych, J-M., et al. (1991)J. Immunol. 141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol.148:3264-3270).

Many human diseases, including chronic infections (such as with humanimmunodeficiency virus (HIV) and tuberculosis) and certain metastaticcarcinomas, also are characterized by a Th1 to Th2 switch (see e.g.,Shearer, G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43;Clerici, M. and Shearer, G. M. (1993) Immunology Today 14:107-111;Yamamura, M., et al. (1993) J. Clin. Invest. 91:1005-1010; Pisa, P., etal. (1992) Proc. Natl. Acad. Sci. USA 89:7708-7712; Fauci, A. S. (1988)Science 239:617-623).

Certain autoimmune diseases have been shown to be associated with apredominant Th1 response. For example, patients with rheumatoidarthritis have predominantly Th1 cells in synovial tissue (Simon, A. K.,et al. (1994) Proc. Natl. Acad. Sci. USA 91:8562-8566) and experimentalautoimmune encephalomyelitis (EAE) can be induced by autoreactive Th1cells (Kuchroo, V. K., et al. (1993) J. Immunol. 151:4371-4381).

The ability to alter or manipulate ratios of Th1 and Th2 subsetsrequires an understanding of the mechanisms by which the differentiationof CD4 T helper precursor cells (Thp), which secrete only IL-2, chooseto become Th1 or Th2 effector cells. It is clear that the cytokinesthemselves are potent Th cell inducers and form an autoregulatory loop(see e.g., Paul, W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder,R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673). Thus, IL4promotes the differentiation of Th2 cells while preventing thedifferentiation of precursors into Th1 cells, while IL-12 and IFN-γ havethe opposite effect.

According to the invention, one way to alter Th1:Th2 ratios is toincrease or decrease the level of selected cytokines by using CD83.Direct administration of cytokines or antibodies to cytokines has beenshown to have an effect on certain diseases mediated by either Th1 orTh2 cells. For example, administration of recombinant IL-4 or antibodiesto IL-12 ameliorate EAE, a Th1-driven autoimmune disease (see Racke; M.K. et al. (1994) J. Exp. Med. 180:1961-1966; and Leonard, J. P. et al.(1995) J. Exp. Med. 181:381-386), while anti-IL-4 antibodies canameliorate the Th2-mediated parasitic disease, Leishmania major (Sadick,M. D. et al. (1990) J. Exp. Med. 171:115-127).

Numerous disease conditions are associated with either a predominantTh1-type response or a predominant Th2-type response and the individualssuffering from such disease conditions could benefit from treatment withthe CD83 related compositions and methods of the invention. Applicationof the immunomodulatory methods of the invention to such diseases isdescribed in further detail below.

Allergies

Allergies are mediated through IgE antibodies whose production isregulated by the activity of Th2 cells and the cytokines producedthereby. In allergic reactions, IL-4 is produced by Th2 cells, whichfurther stimulates production of IgE antibodies and activation of cellsthat mediate allergic reactions, i.e., mast cells and basophils. IL-4also plays an important role in eosinophil mediated inflammatoryreactions.

Accordingly, the stimulation of CD83 production by use of thecompositions and methods of the invention can be used to inhibit theproduction of Th2-associated cytokines, for example IL-4, in allergicpatients as a means to down-regulate production of pathogenic IgEantibodies. A stimulatory agent may be directly administered to thesubject mammal. Alternatively, the CD83 stimulatory agent (e.g. CD83expression cassette) can be administered to cells (e.g., Thp cells orTh2 cells) that may be obtained from the subject and those modifiedcells can be readministered to the subject mammal. Moreover, in certainsituations it may be beneficial to co-administer the allergen togetherwith the stimulatory agent either to the subject or to cells treatedwith the stimulatory agent. Such co-administration can inhibit (e.g.,desensitize) the allergen-specific response. The treatment may befurther enhanced by administering Th1-promoting agents, such as thecytokine IL-12 or antibodies to Th2-associated cytokines (e.g.,anti-IL-4 antibodies), to the allergic subject in amounts sufficient tofurther stimulate a Th1-type response.

Cancer

The invention also relates to CD83-related methods for increasinginterleukin-10 (IL-10) levels to reduce the spread of neoplasticdiseases and/or prevent neoplastic diseases and the growth of a tumor.According to the invention, decreased CD83 activity can dramaticallyincrease the levels of IL-10 in the body and such increasedinterleukin-10 can be used to treat neoplastic diseases. Hence, theinvention provides a method for preventing or treating tumors in amammal, which involves diminishing CD83 expression or activity in themammal. In various embodiments, the tumor is IL-2-dependent, aplasmacytoma, or a leukemia, including a lymphocytic leukemia such as aB cell lymphocytic leukemia.

The invention also provides methods for increasing T cell activation orT cell proliferation by increasing CD83 activity or expression. Suchmethods can also be used to prevent or treat tumors in a mammal.

Infectious Diseases

The expression of Th2-promoting cytokines also has been reported toincrease during a variety of infectious diseases. For example, HIVinfection, tuberculosis, leishmaniasis, schistosomiasis, filarialnematode infection, intestinal nematode infection and other suchinfectious diseases are associated with a Th1 to Th2 shift in the immuneresponse. See e.g., Shearer, G. M. and Clerici, M. (1992) Prog. Chem.Immunol. 54:2143; Clerici, M. and Shearer, G. M. (1993) Immunology Today14:107-111; Fauci, A. S. (1988) Science 239:617-623; Locksley, R. M. andScott, P. (1992) Immunoparasitology Today 1:A58-A61; Pearce, E. J., etal. (1991) J. Exp. Med. 173:159-166; Grzych, J-M., et al. (1991) J.Immunol. 141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol.148:3264-3270; Bancroft, A. J., et al. (1993) J. Immunol. 150:1395-1402;Pearlman, E., et al. (11993) Infect. Immun. 61:1105-1112; Else, K. J.,et al. (1994) J. Exp. Med. 179:347-351.

Accordingly, the stimulatory CD83-related compositions and methods ofthe invention can be used to inhibit the production of Th2-cells insubjects with infectious diseases to promote an ongoing Th1 response inthe patients and to ameliorate the course of the infection. Thetreatment may be further enhanced by administering other Th1-promotingagents, such as the cytokine IL-12 or antibodies to Th2-associatedcytokines (e.g., anti-IL-4 antibodies), to the recipient in amountssufficient to further stimulate a Th 1-type response.

Hence, for example, infections of the following microbial organisms canbe treated by the methods of the invention: Aeromonas spp., Bacillusspp., Bacteroides spp., Campylobacter spp., Clostridium spp.,Enterobacter spp., Enterococcus spp., Escherichia spp., Gastrospirillumsp., Helicobacter spp., Klebsiella spp., Salmonella spp., Shigella spp.,Staphylococcus spp., Pseudomonas spp., Vibrio spp., Yersinia spp., andthe like. Infections that can be treated by the methods of the inventioninclude those associated with staph infections (Staphylococcus aureus),typhus (Salmonella typhi), food poisoning (Escherichia coli, such asO157:H7), bascillary dysentery (Shigella dysenteria), pneumonia(Psuedomonas aerugenosa and/or Pseudomonas cepacia), cholera (Vivriocholerae), ulcers (Helicobacter pylori) and others. E. coli serotypeO157:H7 has been implicated in the pathogenesis of diarrhea, hemorrhagiccolitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenicpurpura (TTP). The methods of the invention are also active againstdrug-resistant and multiply-drug resistant strains of bacteria, forexample, multiply-resistant strains of Staphylococcus aureus andvancomycin-resistant strains of Enterococcus faecium and Enterococcusfaecalis.

The methods of the invention are also effective against viruses. Theterm “virus” refers to DNA and RNA viruses, viroids, and prions. Virusesinclude both enveloped and non-enveloped viruses, for example, hepatitisA virus, hepatitis B virus, hepatitis C virus, human immunodeficiencyvirus (HIV), poxviruses, herpes viruses, adenoviruses, papovaviruses,parvoviruses, reoviruses, orbiviruses, picornaviruses, rotaviruses,alphaviruses, rubivirues, influenza virus type A and B, flaviviruses,coronaviruses, paramyxoviruses, morbilliviruses, pneumoviruses,rhabdoviruses, lyssaviruses, orthmyxoviruses, bunyaviruses,phleboviruses, nairoviruses, hepadnaviruses, arenaviruses, retroviruses,enteroviruses, rhinoviruses and the filovirus.

Autoimmune Diseases

The CD83-related compositions and methods of the invention can be usedin the treatment of autoimmune diseases that are associated with aTh2-type dysfunction. Many autoimmune disorders are the result ofinappropriate activation of T cells that are reactive against “selftissues” and that promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Modulation of T helper-typeresponses can have an effect on the course of the autoimmune disease.For example, in experimental allergic encephalomyelitis, stimulation ofa Th2-type response by administration of IL-4 at the time of theinduction of the disease diminishes the intensity of the autoimmunedisease (Paul, W. E., et al. (1994) Cell 76:241-251). Furthermore,recovery of the animals from the disease has been shown to be associatedwith an increase in a Th2-type response as evidenced by an increase ofTh2-specific cytokines (Koury, S. J., et al. (1992) J. Exp. Med.176:1355-1364). Moreover, T cells that can suppress EAE secreteTh2-specific cytokines (Chen, C., et al. (1994) Immunity 1:147-154).Since stimulation of a Th2-type response in experimental allergicencephalomyelitis has a protective effect against the disease,stimulation of a Th2 response in subjects with multiple sclerosis (forwhich EAE is a model) is likely to be beneficial therapeutically.

Similarly, stimulation of a Th2-type response in type I diabetes in miceprovides a protective effect against the disease. Indeed, treatment ofNOD mice with IL-4 (which promotes a Th2 response) prevents or delaysonset of type I diabetes that normally develops in these mice (Rapoport,M. J., et al. (1993) J. Exp. Med. 178:87-99). Thus, inhibition of CD83production can stimulate IL-4 production and/or a Th2 response in asubject suffering from or susceptible to diabetes may ameliorate theeffects of the disease or inhibit the onset of the disease.

Yet another autoimmune disease in which stimulation of a Th2-typeresponse may be beneficial is rheumatoid arthritis (RA). Studies haveshown that patients with rheumatoid arthritis have predominantly Th1cells in synovial tissue (Simon, A. K., et al., (1994) Proc. Natl. Acad.Sci. USA 91:8562-8566). By stimulating a Th2 response in a subject withrheumatoid arthritis, the detrimental Th1 response can be concomitantlydown-modulated to thereby ameliorate the effects of the disease.

Accordingly, the CD83-related compositions and methods of the inventioncan be used to stimulate production of Th2-associated cytokines insubjects suffering from, or susceptible to, an autoimmune disease inwhich a Th2-type response is beneficial to the course of the disease.Such compositions and methods would modulate CD83 activity. In someembodiments, the compositions would decrease CD83 activity and therebyincrease the level of certain cytokines, for example, IL-4 levels areincreased when CD83 activity is diminished. The treatment may be furtherenhanced by administering other Th2-promoting agents, such as IL-4itself or antibodies to Th1-associated cytokines, to the subject inamounts sufficient to further stimulate a Th2-type response. Thetreatment may be further enhanced by administering a Th1-promotingcytokine (e.g., IFN-γ) to the subject in amounts sufficient to furtherstimulate a Th1-type response.

The efficacy of CD83-related for treating autoimmune diseases can betested in the animal models provided herein or other models of humandiseases (e.g., EAE as a model of multiple sclerosis and the NOD mice asa model for diabetes). Such animal models include the mrl/lpr/lpr mouseas a model for lupus erythematosus, murine collagen-induced arthritis asa model for rheumatoid arthritis, and murine experimental myastheniagravis (see Paul ed., Fundamental Immunology, Raven Press, New York,1989, pp. 840-856). A CD83-modulatory (i.e., stimulatory or inhibitory)agent of the invention is administered to test animals and the course ofthe disease in the test animals is then monitored by the standardmethods for the particular model being used. Effectiveness of themodulatory agent is evidenced by amelioration of the disease conditionin animals treated with the agent as compared to untreated animals (oranimals treated with a control agent).

Non-limiting examples of autoimmune diseases and disorders having anautoimmune component that may be treated according to the inventioninclude diabetes mellitus, arthritis (including rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis),multiple sclerosis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, includingkeratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopeciaareata, allergic responses due to arthropod bite reactions, Crohn'sdisease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Crohn's disease, Graves opthalmopathy, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.

Transplantation

While graft rejection or graft acceptance may not be attributableexclusively to the action of a particular T cell subset (i.e., Th1 orTh2 cells) in the graft recipient, studies have implicated a predominantTh2 response in prolonged graft survival and a predominant Th1 responsein graft rejection (for a discussion see Dallman, M. J. (1995) Curr.Opin. Immunol. 7:632-638; Takeuchi, T. et al. (1992) Transplantation53:1281-1291; Tzakis, A. G. et al. (1994) J. Pediatr. Surg. 29:754-756;That, N. L. et al. (1995) Transplantation 59:274-281. Additionally,adoptive transfer of cells having a Th2 cytokine phenotype prolongs skingraft survival (Maeda, H. et al. (1994) Int. Immunol. 6:855-862) andreduces graft-versus-host disease (Fowler, D. H. et al. (1994) Blood84:3540-3549; Fowler, D. H. et al. (1994) Prog. Clin. Biol. Res.389:533-540). Furthermore, administration of IL-4, which promotes Th2differentiation, prolongs cardiac allograft survival (Levy, A. E. andAlexander, J. W. (1995) Transplantation 60:405-406), whereasadministration of IL-12 in combination with anti-IL-10 antibodies, whichpromotes Th1 differentiation, enhances skin allograft rejection(Gorczynski, R. M. et al. (1995) Transplantation 60:1337-1341).

As provided herein, loss of CD83 function increases interleukin-4production, which in turn promotes the differentiation of Th2 cells anddepresses the differentiation of precursor cells into Th1 cells.Accordingly, methods of the invention that involve decreasing CD83function can be used to stimulate production of Th2-associated cytokinesin transplant recipients to prolong survival of the graft. These methodscan be used both in solid organ transplantation and in bone marrowtransplantation (e.g., to inhibit graft-versus-host disease). Thesemethods can involve either direct administration of a CD83 inhibitoryagent to the transplant recipient or ex vivo treatment of cells obtainedfrom the subject (e.g., Thp, Th1 cells, B cells, non-lymphoid cells)with an inhibitory agent followed by readministration of the cells tothe subject. The treatment may be further enhanced by administeringother Th2-promoting agents, such as IL-4 itself or antibodies toTh1-associated cytokines, to the recipient in amounts sufficient tofurther stimulate a Th2-type response.

Additional Methods of Using CD83

In addition to the foregoing disease situations, the modulatory methodsof the invention also are useful for other purposes.

For example, inhibition of CD83 activity or function gives rise toincreased granulocyte macrophage-colony stimulating factor (GM-CSF).Granulocyte macrophage colony stimulating factor is a hematopoieticgrowth factor that promotes the proliferation and differentiation ofhematopoietic progenitor cells. GM-CSF is approved for treatment ofpatients requiring increased proliferation of white blood cells. Dataindicates that GM-CSP is also useful as a vaccine adjuvant Morrissey, etal., J. Immunology 139, 1113-1119 (1987). GM-CSF can also be used totreat patients prone to infection such as those undergoing high riskbowel surgery, trauma victims and individuals with HIV.

Accordingly, the invention provides a method of increasing the levels ofGM-CSF in a mammal or in a mammalian cell by administering an agent thatmodulates or inhibits CD83 activity or expression.

The invention also provides a method of decreasing the levels of GM-CSFin a mammal or in a mammalian cell by administering an agent thatmodulates or stimulates CD83 activity or expression.

Moreover, in other embodiments the CD83 inhibitory methods of theinvention can be used to stimulate production of IL-4 or IL-10 in vitrofor commercial production of these cytokines. For example, CD4+ T cellswith a null or other mutation in the CD83 gene can be cultured and thenstimulated to produce cytokines, for example, by use of anti-CD3 and/oranti-CD28 antibodies to activate the mutant CD4+ T cells. Significantamounts of IL-4 and IL-10 can then be isolated from the culture media.Alternatively, CD4+ T cells can be contacted with the CD83 inhibitoryagent in vitro to stimulate IL-4 or IL-10 production and the IL-4 orIL-10 can be recovered from the culture supernatant. The isolated IL-4and/or IL-10 can be further purified if necessary, and packaged forcommercial use.

The methods of the invention can be adapted to vaccinations to promoteeither a Th1 or a Th2 response to an antigen of interest in a subject.That is, CD83 or CD83 modulators of the invention can serve as adjuvantsto direct an immune response to a vaccine either to a Th1 response or aTh2 response. For example, to stimulate an antibody response to anantigen of interest (i.e., for vaccination purposes), the antigen and aCD83 inhibitory agent of the invention can be coadministered to asubject to promote a Th2 response to the antigen in the subject, sinceTh2 responses provide efficient B cell help and promote IgG1 production.

Alternatively, to promote a cellular immune response to an antigen ofinterest, the antigen and a CD83 stimulating agent of the invention canbe coadministered to a subject to promote a Th1 response to the antigenin a subject, since Th1 responses favor the development of cell-mediatedimmune responses (e.g., delayed hypersensitivity responses).

The antigen of interest and the modulatory agent can be formulatedtogether into a single pharmaceutical composition or in separatecompositions.

Thus, in some embodiments, the antigen of interest and the modulatoryagent are administered simultaneously to the subject. Alternatively, incertain situations it may be desirable to administer the antigen firstand then the modulatory agent or vice versa. For example, in the case ofan antigen that naturally evokes a Th1 response, it may be beneficial tofirst administer the antigen alone to stimulate a Th1 response and thenadminister a CD83 inhibitory agent, alone or together with a boost ofantigen, to shift the immune response to a Th2 response.

According to the invention, any agent that can modulate CD83 to increaseor decrease cytokine levels, increase or decrease T cell levels orproduce any other CD83-related response can be used in the compositionsand methods of the invention. In some embodiments, anti-CD83 antibodiesof the invention are used to either activate or inhibit CD83 activity.Activation or inhibition by such antibodies can depend on the epitope towhich the antibody binds. Hence, antibodies may play a role in boostingor depressing CD83 activity. These CD83 modulatory agents, includinganti-CD83 antibodies, are described in more detail below.

Stimulating or Inhibiting CD83

According to the invention, any agent that can stimulate CD83 to performits natural functions can be used in the compositions and methods of theinvention as a CD83 stimulatory agent. Indicators that CD83 activity isstimulated include increased IL-2 cytokine levels, increased T celllevels, and increased TNF levels relative to unstimulated levels in wildtype CD83 cells. Examples of CD83 stimulatory agents include, forexample, the CD83 gene product itself, certain anti-CD83 antibodies,CD83-encoding nucleic acids (DNA or RNA), factors that promote CD83transcription or translation, organic molecules, peptides and the like.

Also, according to the invention, any agent that can inhibit CD83 fromperforming its natural functions can be used in the compositions andmethods of the invention as a CD83 inhibitory agent. Indicators thatCD83 activity is inhibited include increased IL-4 cytokine levels,increased IL-10 levels, decreased IL-2 production, decreased T celllevels, and decreased TNF levels relative to uninhibited levels in wildtype CD83 cells.

Examples of CD83 inhibitors include anti-CD83 antibodies, CD83anti-sense nucleic acids (e.g. nucleic acids that can hybridize to CD83nucleic acids), organic compounds, peptides and agents that can mutatean endogenous CD83 gene. In some embodiments, the CD83 stimulatory orinhibitory agents are proteins, for example, CD83 gene products,anti-CD83 antibody preparations, CD83 inhibitors, peptides and proteinfactors that can promote CD83 transcription or translation. In otherembodiments, the CD83 stimulatory or inhibitory agents are peptides ororganic molecules. Such proteins, organic molecules and organicmolecules can be prepared and/or purified as described herein or bymethods available in the art, and administered as provided herein.

In other embodiments, the CD83 stimulatory or inhibitory agents can benucleic acids including recombinant expression vectors or expressioncassettes encoding CD83 gene products, CD83 transcription factors, CD83anti-sense nucleic acid, intracellular antibodies capable of binding toCD83 or dominant negative CD83 inhibitors. Such nucleic acids can beoperably linked to a promoter that is functional in a mammalian cell,and then introduced into cells of the subject mammal using methods knownin the art for introducing nucleic acid (e.g., DNA) into cells.

The “promoter functional in a mammalian cell” or “mammalian promoter” iscapable of directing transcription of a polypeptide coding sequenceoperably linked to the promoter. The promoter should generally be activein T cells and antigen presenting cells and may be obtained from a genethat is expressed in T cells or antigen presenting cells. However, itneed not be a T cell-specific or an antigen presenting cellspecific-promoter. Instead, the promoter may be selected from anymammalian or viral promoter that can function in a T cell. Hence thepromoter may be an actin promoter, an immunoglobulin promoter, aheat-shock promoter, or a viral promoter obtained from the genome ofviruses such as adenoviruses, retroviruses, lentiviruses, herpesviruses, including but not limited to, polyoma virus, fowlpox virus,adenovirus 2, bovine papilloma virus, avian sarcoma virus,cytomegalovirus (CMV), hepatitis-B virus, Simian Virus 40 (SV40),Epstein Barr virus (EBV), feline immunodeficiency virus (FIV), andSr.alpha., or are respiratory synsitial viral promoters (RSV) or longterminal repeats (LTRs) of a retrovirus, i.e., a Moloney Murine LeukemiaVirus (MoMuLv) (Cepko et al. (1984) Cell 37:1053-1062). The promoterfunctional in a mammalian cell can be inducible or constitutive.

Any cloning procedure used by one of skill in the art can be employed tomake the expression vectors or expression that comprise a promoteroperably linked to a CD83 nucleic acid, CD83 transcription factor or anucleic acid encoding an anti-CD83 antibody. See, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., 1989; Sambrook et al., Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, N.Y., 2001.

After constructing an expression vector or an expression cassetteencoding CD83 gene products, CD83 transcription factors, CD83 anti-sensenucleic acid, intracellular antibodies capable of binding to CD83 ordominant negative CD83 inhibitors, mammalian cells can be transformedwith the vector or cassette. Examples of such methods include:

Direct Injection: Naked DNA can be introduced into cells in vivo bydirectly injecting the DNA into the cells (see e.g., Acsadi et al.(1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468).For example, a delivery apparatus (e.g., a “gene gun”) for injecting DNAinto cells in vivo can be used. Such an apparatus is commerciallyavailable (e.g., from BioRad).

Receptor-Mediated DNA Uptake: Naked DNA can also be introduced intocells in vivo by complexing the DNA to a cation, such as polylysine,which is coupled to a ligand for a cell-surface receptor (see forexample Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson etal. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320).Binding of the DNA-ligand complex to the receptor facilitates uptake ofthe DNA by receptor-mediated endocytosis. A DNA-ligand complex linked toadenovirus capsids that naturally disrupt endosomes, thereby releasingmaterial into the cytoplasm can be used to avoid degradation of thecomplex by intracellular lysosomes (see for example Curiel et al. (1991)Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.Acad. Sci. USA 90:2122-2126).

Retroviruses: Defective retroviruses are well characterized for use ingene transfer for gene therapy purposes (for a review see Miller, A. D.(1990) Blood 76:271). A recombinant retrovirus can be constructed havingnucleotide sequences of interest incorporated into the retroviralgenome. Additionally, portions of the retroviral genome can be removedto render the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions that can be used toinfect a target cell through the use of a helper virus by standardtechniques. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14 and otherstandard laboratory manuals. Examples of suitable retroviruses includepLJ, pZIP, pWE and pEM which are available to those skilled in the art.Examples of suitable packaging virus lines include ΨCrip, ΨCre, Ψ2 andΨAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including epithelial cells, endothelialcells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitroand/or in vivo (see for example Eglitis, et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; andPCT Application WO 92/07573). Retroviral vectors require target celldivision in order for the retroviral genome (and foreign nucleic acidinserted into it) to be integrated into the host genome to stablyintroduce nucleic acid into the cell. Thus, it may be necessary tostimulate replication of the target cell.

Adenoviruses: The genome of an adenovirus can be manipulated such thatit encodes and expresses a gene product of interest but is inactivatedin terms of its ability to replicate in a normal lytic viral life cycle.See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeldet al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are available to those skilled in the art. Recombinantadenoviruses are advantageous in that they do not require dividing cellsto be effective gene delivery vehicles and can be used to infect a widevariety of cell types, including airway epithelium (Rosenfeld et al.(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc.Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin etal. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use are deletedfor all or parts of the viral E1 and E3 genes but retain as much as 80%of the adenoviral genetic material.

Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

Transformed mammalian cells can then be identified and administered tothe mammal from whence they came to permit expression of a CD83 geneproduct, CD83 transcription factor, CD83 anti-sense nucleic acid,intracellular antibody capable of binding to CD83 proteins, or dominantnegative CD83 inhibitors. The efficacy of a particular expression vectorsystem and method of introducing nucleic acid into a cell can beassessed by standard approaches routinely used in the art. For example,DNA introduced into a cell can be detected by a filter hybridizationtechnique (e.g., Southern blotting). RNA produced by transcription of anintroduced DNA can be detected, for example, by Northern blotting, RNaseprotection or reverse transcriptase-polymerase chain reaction (RT-PCR).The CD83 gene product can be detected by an appropriate assay, forexample, by immunological detection of a produced CD83 protein, such aswith a CD83-specific antibody.

CD83 Antibodies

The invention provides antibody preparations directed against the mutantand wild type CD83 polypeptides of the invention, for example, against apolypeptide having SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8 orSEQ ID NO:9. Other antibodies of interest can bind to the cytoplasmictail of CD83.

In one embodiment, the invention provides antibodies that block thefunction of CD83 polypeptides. Such antibodies may be used as CD83inhibitory agents in the methods of the invention as described herein.In another embodiment, the antibodies of the invention can activate CD83activity. Such activating antibodies may be used as CD83 stimulatoryagents.

All antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an antigen binding region known as avariable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Clothia et al., J. Mol.Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA82, 4592-4596 (1985).

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and several of these may be further divided into subclasses(isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. Theheavy chains constant domains that correspond to the different classesof immunoglobulins are called alpha (α), delta (δ), epsilon (ε), gamma(γ) and mu (μ), respectively. The light chains of antibodies can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino sequences of their constant domain. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies. The variable domains are forbinding and determine the specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) also known as hypervariable regions both in the lightchain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four FR regions, largely adopting a β-sheet configuration,connected by three CDRs, which form loops connecting, and in some casesforming part of, the β-sheet structure. The CDRs in each chain are heldtogether in close proximity by the FR regions and, with the CDRs fromthe other chain, contribute to the formation of the antigen binding siteof antibodies. The constant domains are not involved directly in bindingan antibody to an antigen, but exhibit various effector function, suchas participation of the antibody in antibody-dependent cellulartoxicity.

An antibody that is contemplated for use in the present invention thuscan be in any of a variety of forms, including a whole immunoglobulin,an antibody fragment such as Fv, Fab, and similar fragments, a singlechain antibody that includes the variable domain complementaritydetermining regions (CDR), and the like forms, all of which fall underthe broad term “antibody,” as used herein. The present inventioncontemplates the use of any specificity of an antibody, polyclonal ormonoclonal, and is not limited to antibodies that recognize andimmunoreact with a specific antigen. In preferred embodiments, in thecontext of both the therapeutic and screening methods described below,an antibody or fragment thereof is used that is immunospecific for anantigen or epitope of the invention.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments, which are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

Antibody fragments retain some ability to selectively bind with itsantigen or receptor and are defined as follows:

(1) Fab is the fragment that contains a monovalent antigen-bindingfragment of an antibody molecule. A Fab fragment can be produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain.

(2) Fab′ is the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain. Two Fab′ fragmentsare obtained per antibody molecule. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region.

(3) (Fab′)₂ is the fragment of an antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction. F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)-V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule. Such single chain antibodiesare also referred to as “single-chain Fv” or “sFv” antibody fragments.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains that enables the sFv to form the desiredstructure for antigen binding. For a review of sFv see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, N.Y., pp. 269-315 (1994).

The term “diabodies” refers to a small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993).

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green, et al., Production of PolyclonalAntisera, in: Immunochemical Protocols (Manson, ed.), pages 1-5 (HumanaPress); Coligan, et al., Production of Polyclonal Antisera in Rabbits,Rats Mice and Hamsters, in: Current Protocols in Immunology, section2.4.1 (1992), which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature, 256:495 (1975); Coligan, et al.,sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: A LaboratoryManual, page 726 (Cold Spring Harbor Pub. (1988)), which are herebyincorporated by reference. Methods of in vitro and in vivo manipulationof monoclonal antibodies are also available to those skilled in the art.For example, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler and Milstein, Nature 256, 495 (1975), or may be made byrecombinant methods, e.g., as described in U.S. Pat. No. 4,816,567. Themonoclonal antibodies for use with the present invention may also beisolated from antibody libraries using the techniques described inClackson et al. Nature 352: 624-628 (1991), as well as in Marks et al.,J. Mol. Biol. 222: 581-597 (1991).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See,e.g., Coligan, et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;Barnes, et al., Purification of Immunoglobulin G (IgG), in: Methods inMolecular Biology, Vol. 10, pages 79-104 (Humana Press (1992).

Another method for generating antibodies involves a Selected LymphocyteAntibody Method (SLAM). The SLAM technology permits the generation,isolation and manipulation of monoclonal antibodies without the processof hybridoma generation. The methodology principally involves the growthof antibody forming cells, the physical selection of specificallyselected antibody forming cells, the isolation of the genes encoding theantibody and the subsequent cloning and expression of those genes.

More specifically, an animal (rabbit, mouse, rat, other) is immunizedwith a source of specific antigen. This immunization may consist ofpurified protein, in either native or recombinant form, peptides, DNAencoding the protein of interest or cells expressing the protein ofinterest. After a suitable period, during which antibodies can bedetected in the serum of the animal (usually weeks to months), blood (orother tissue) from the animal is harvested. Lymphocytes are isolatedfrom the blood and cultured under specific conditions to generateantibody-forming cells, with antibody being secreted into the culturemedium. These cells are detected by any of several means (complementmediated lysis of antigen-bearing cells, fluorescence detection orother) and then isolated using micromanipulation technology. Theindividual antibody forming cells are then processed for eventual singlecell PCR to obtain the expressed Heavy and Light chain genes that encodethe specific antibody. Once obtained and sequenced, these genes arecloned into an appropriate expression vector and recombinant, monoclonalantibody produced in a heterologous cell system. These antibodies arethen purified via standard methodologies such as the use of protein Aaffinity columns. These types of methods are further described inBabcook, et al., Proc. Natl. Acad. Sci. (USA) 93: 7843-7848 (1996); U.S.Pat. No. 5,627,052; and PCT WO 92/02551 by Schrader.

Another method involves humanizing a monoclonal antibody by recombinantmeans to generate antibodies containing human specific and recognizablesequences. See, for review, Holmes, et al., J. Immunol., 158:2192-2201(1997) and Vaswani, et al., Annals Allergy, Asthma & Immunol.,81:105-115 (1998). The term “monoclonal antibody” as used herein refersto an antibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the antibody is obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567);Morrison et al. Proc. Natl. Acad. Sci. 81, 6851-6855 (1984).

Methods of making antibody fragments are also known in the art (see forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, (1988), incorporated herein by reference).Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofDNA encoding the fragment. Antibody fragments can be obtained by pepsinor papain digestion of whole antibodies conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab=monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly. These methodsare described, for example, in U.S. Pat. No. 4,036,945 and No.4,331,647, and references contained therein. These patents are herebyincorporated in their entireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of V_(H) and V_(L) chains. This association may benoncovalent or the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde.Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The structural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Methods for producing sFvsare described, for example, by Whitlow, et al., Methods: a Companion toMethods in Enzymology, Vol. 2, page 97 (1991); Bird, et al., Science242:423-426 (1988); Ladner, et al, U.S. Pat. No. 4,946,778; and Pack, etal., Bio/Technology 11:1271-77 (1993).

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick, et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 106 (1991).

The invention further contemplates human and humanized forms ofnon-human (e.g. murine) antibodies. Such humanized antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a nonhuman species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity.

In some instances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, humanized antibodies can comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the Fv regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., Nature 321,522-525 (1986); Reichmann et al., Nature 332, 323-329 (1988); Presta,Curr. Op. Struct. Biol. 2, 593-596 (1992); Holmes, et al., J. Immunol.,158:2192-2201 (1997) and Vaswani, et al., Annals Allergy, Asthma &Immunol., 81:105-115 (1998).

The invention also provides methods of mutating antibodies to optimizetheir affinity, selectivity, binding strength or other desirableproperty. A mutant antibody refers to an amino acid sequence variant ofan antibody. In general, one or more of the amino acid residues in themutant antibody is different from what is present in the referenceantibody. Such mutant antibodies necessarily have less than 100%sequence identity or similarity with the reference amino acid sequence.In general, mutant antibodies have at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the reference antibody. Preferably,mutant antibodies have at least 80%, more preferably at least 85%, evenmore preferably at least 90%, and most preferably at least 95% aminoacid sequence identity or similarity with the amino acid sequence ofeither the heavy or light chain variable domain of the referenceantibody.

The antibodies of the invention are isolated antibodies. An isolatedantibody is one that has been identified and separated and/or recoveredfrom a component of the environment in which it was produced.Contaminant components of its production environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. The term “isolated antibody” also includesantibodies within recombinant cells because at least one component ofthe antibody's natural environment will not be present. Ordinarily,however, isolated antibody will be prepared by at least one purificationstep.

If desired, the antibodies of the invention can be purified by anyavailable procedure. For example, the antibodies can be affinitypurified by binding an antibody preparation to a solid support to whichthe antigen used to raise the antibodies is bound. After washing offcontaminants, the antibody can be eluted by known procedures. Those ofskill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (see for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991, incorporated by reference).

In preferred embodiments, the antibody will be purified as measurable byat least three different methods: 1) to greater than 95% by weight ofantibody as determined by the Lowry method, and most preferably morethan 99% by weight; 2) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequentator; or 3) to homogeneity by SDS-PAGE underreducing or non-reducing conditions using Coomasie blue or, preferably,silver stain.

The invention also provides antibodies that can bind to CD83polypeptides. Sequences of complementarity determining regions (CDRs) orhypervariable regions from light and heavy chains of these anti-CD83antibodies are provided. For example, a heavy chain variable regionhaving a CDR1 sequence of SYDMT (SEQ ID NO:23), SYDMS (SEQ ID NO:24),DYDLS (SEQ ID NO:25) or SYDMS (SEQ ID NO:26) can be used in an antibodyor other binding moiety to bind to CD83 gene products. In otherembodiments, a heavy chain variable region having a CDR2 sequence ofYASGSTYY (SEQ ID NO:27), SSSGTTYY (SEQ ID NO:28), YASGSTYY (SEQ IDNO:29), AIDGNPYY (SEQ ID NO:30) or STAYNSHY (SEQ ID NO:31) can be usedin an antibody or other binding moiety to bind to CD83 gene products. Infurther embodiments of the invention, a heavy chain variable regionhaving a CDR3 sequence of EHAGYSGDTGH (SEQ ID NO:32), EGAGVSMT (SEQ IDNO:33), EDAGFSNA (SEQ ID NO:34), GAGD (SEQ ID NO:35) or GGSWLD (SEQ IDNO:36) can be used in an antibody or other binding moiety to bind toCD83 gene products.

Moreover, a light chain variable region having a CDR1 sequence of RCAYD(SEQ ID NO:37), RCADVV (SEQ ID NO:38), or RCALV (SEQ ID NO:39) can beused in an antibody or other binding moiety to bind to CD83 geneproducts. In other embodiments, a light chain variable region having aCDR2 sequence of QSISTY (SEQ ID NO:40), QSVSSY (SEQ ID NO:41), ESISNY(SEQ ID NO:42), KNVYNNNW (SEQ ID NO:43), or QSVYDNDE (SEQ ID NO:43) canbe used in an antibody or other binding moiety to bind to CD83 geneproducts. In further embodiments, a light chain variable region having aCDR3 sequence of QQGYTHSNVDNV (SEQ ID NO:44), QQGYSISDIDNA (SEQ IDNO:45), QCTSGGKFISDGAA (SEQ ID NO:46), AGDYSSSSDNG (SEQ ID NO:47), orQATHYSSDWLTY (SEQ ID NO:48) can be used in an antibody or other bindingmoiety to bind to CD83 gene products.

Light and heavy chains that can bind CD83 polypeptides are also providedby the invention. For example, in one embodiment, the invention providesa 20D04 light chain that can bind to CD83 polypeptides. The amino acidsequence for this 20D04 light chain is provided below (SEQ ID NO:11).

  1 MDMRAPTQLL GLLLLWLPGA RCADVVMTQT PASVSAAVGG  41 TVTINCQASESISNYLSWYQ QKPGQPPKLL IYRTSTLASG  81 VSSRFKGSGS GTEYTLTISG VQCDDVATYYCQCTSGGKFI 121 SDGAAFGGGT EVVVKGDPVA PTVLLFPPSS DEVATGTVTI 161VCVANKYFPD VTVTWEVDGT TQTTGIENSK TPQNSADCTY 201 NLSSTLTLTS TQYNSHKEYTCKVTQGTTSV VQSFSRKNC

A nucleic acid sequence for this 20D04 anti-CD83 light chain is providedbelow (SEQ ID NO:12).

  1 ATGGACATGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC  41 TGCTCTGGCTCCCAGGTGCC AGATGTGCCG ATGTCGTGAT  81 GACCCAGACT CCAGCCTCCG TGTCTGCAGCTGTGGGAGGC 121 ACAGTCACCA TCAATTGCCA GGCCAGTGAA AGCATTAGCA 161ACTACTTATC CTGGTATCAG CAGAAACCAG GGCAGCCTCC 201 CAAGCTCCTG ATCTACAGGACATCCACTCT GGCATCTGGG 241 GTCTCATCGC GGTTCAAAGG CAGTGGATCT GGGACAGAGT281 ACACTCTCAC CATCAGCGGC GTGCAGTGTG ACGATGTTGC 321 CACTTACTACTGTCAATGCA CTTCTGGTGG GAAGTTCATT 361 AGTGATGGTG CTGCTTTCGG CGGAGGGACCGAGGTGGTGG 401 TCAAAGGTGA TCCAGTTGCA CCTACTGTCC TCCTCTTCCC 441ACCATCTAGC GATGAGGTGG CAACTGGAAC AGTCACCATC 481 GTGTGTGTGG CGAATAAATACTTTCCCGAT GTCACCGTCA 521 CCTGGGAGGT GGATGGCACC ACCCAAACAA CTGGCATCGA561 GAACAGTAAA ACACCGCAGA ATTCTGCAGA TTGTACCTAC 601 AACCTCAGCAGCACTCTGAC ACTGACCAGC ACACAGTACA 641 ACAGCCACAA AGAGTACACC TGCAAGGTGACCCAGGGCAC 681 GACCTCAGTC GTCCAGAGCT TCAGTAGGAA GAACTGTTAA

In another embodiment, the invention provides a 20D04 heavy chain thatcan bind to CD83 polypeptides. The amino acid sequence for this 20D04heavy chain is provided below (SEQ ID NO:13).

  1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC  41 TVSGFSLSNNAINWVRQAPG KGLEWIGYIW SGGLTYYANW  81 AEGRFTISKT STTVDLKMTS PTIEDTATYFCARGINNSAL 121 WGPGTLVTVS SGQPKAPSVF PLAPCCGDTP SSTVTLGCLV 161KGYLEPEVTV TWNSGTLTNG VRTFPSVRQS SGLYSLSSVV 201 SVTSSSQPVT CNVAHPATNTKVDKTVAPST CSKPTCPPPE 241 LLGGPSVFIF PPKPKDTLMI SRTPEVTCVV VDVSQDDPEV281 QFTWYINNEQ VRTARPPLRE QQFNSTIRVV STLPIAHQDW 321 LRGKEFKCKVHNKALPAPIE KTISKARGQP LEPKVYTMGP 361 PREELSSRSV SLTCMINGFY PSDISVEWEKNGKAEDNYKT 401 TPAVLDSDGS YFLYNKLSVP TSEWQRGDVF TCSVMHEALH 441NHYTQKSISR SPGK

A nucleic acid sequence for this 20D04 anti-CD83 heavy chain is providedbelow (SEQ ID NO:14).

   1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC   41 TCAAAGGTGTCCAGTGTCAG TCGGTGGAGG AGTCCGGGGG   81 TCGCCTGGTC ACGCCTGGGA CACCCCTGACACTCACCTGC  121 ACCGTCTCTG GATTCTCCCT CAGTAACAAT GCAATAAACT  161GGGTCCGCCA GGCTCCAGGG AAGGGGCTAG AGTGGATCGG  201 ATACATTTGG AGTGGTGGGCTTACATACTA CGCGAACTGG  241 GCGGAAGGCC GATTCACCAT CTCCAAAACC TCGACTACGG 281 TGGATCTGAA GATGACCAGT CCGACAATCG AGGACACGGC  321 CACCTATTTCTGTGCCAGAG GGATTAATAA CTCCGCTTTG  361 TGGGGCCCAG GCACCCTGGT CACCGTCTCCTCAGGGCAAC  401 CTAAGGCTCC ATCAGTCTTC CCACTGGCCC CCTGCTGCGG  441GGACACACCC TCTAGCACGG TGACCTTGGG CTGCCTGGTC  481 AAAGGCTACC TCCCGGAGCCAGTGACCGTG ACCTGGAACT  521 CGGGCACCCT CACCAATGGG GTACGCACCT TCCCGTCCGT 561 CCGGCAGTCC TCAGGCCTCT ACTCGCTGAG CAGCGTGGTG  601 AGCGTGACCTCAAGCAGCCA GCCCGTCACC TGCAACGTGG  641 CCCACCCAGC CACCAACACC AAAGTGGACAAGACCGTTGC  681 GCCCTCGACA TGCAGCAAGC CCACGTGCCC ACCCCCTGAA  721CTCCTGGGGG GACCGTCTGT CTTCATCTTC CCCCCAAAAC  761 CCAAGGACAC CCTCATGATCTCACGCACCC CCGAGGTCAC  801 ATGCGTGGTG GTGGACGTGA GCCAGGATGA CCCCGAGGTG 841 CAGTTCACAT GGTACATAAA CAACGAGCAG GTGCGCACCG  881 CCCGGCCGCCGCTACGGGAG CAGCAGTTCA ACAGCACGAT  921 CCGCGTGGTC AGCACCCTCC CCATCGCGCACCAGGACTGG  961 CTGAGGGGCA AGGAGTTCAA GTGCAAAGTC CACAACAAGG 1001CACTCCCGGC CCCCATCGAG AAAACCATCT CCAAAGCCAG 1041 AGGGCAGCCC CTGGAGCCGAAGGTCTACAC CATGGGCCCT 1081 CCCCGGGAGG AGCTGAGCAG CAGGTCGGTC AGCCTGACCT1121 GCATGATCAA CGGCTTCTAC CCTTCCGACA TCTCGGTGGA 1161 GTGGGAGAAGAACGGGAAGG CAGAGGACAA CTACAAGACC 1201 ACGCCGGCCG TGCTGGACAG CGACGGCTCCTACTTCCTCT 1241 ACAACAAGCT CTCAGTGCCC ACGAGTGAGT GGCAGCGGGG 1281CGACGTCTTC ACCTGCTCCG TGATGCACGA GGCCTTGCAC 1321 AACCACTACA CGCAGAAGTCCATCTCCCGC TCTCCGGGTA 1361 AA

In another embodiment, the invention provides a 11G05 light chain thatcan bind to CD83 polypeptides. The amino acid sequence for this 11G05light chain is provided below (SEQ ID NO:15).

  1 MDTRAPTQLL GLLLLWLPGA RCADVVMTQT PASVSAAVGG  41 TVTINCQSSKNVYNNNWLSW FQQKPGQPPK LLIYYASTLA  81 SGVPSRFRGS GSGTQFTLTI SDVQCDDAATYYCAGDYSSS 121 SDNGFGGGTE VVVKGDPVAP TVLLFPPSSD EVATGTVTIV 161CVANKYFPDV TVTWEVDGTT QTTGIENSKT PQNSADCTYN 201 LSSTLTLTST QYNSHKEYTCKVTQGTTSVV QSFSRKNC

A nucleic acid sequence for this 11G05 anti-CD83 light chain is providedbelow (SEQ ID NO:16).

  1 ATGGACACCA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC  41 TGCTCTGGCTCCCAGGTGCC AGATGTGCCG ACGTCGTGAT  81 GACCCAGACT CCAGCCTCCG TGTCTGCAGCTGTGGGAGGC 121 ACAGTCACCA TCAATTGCCA GTCCAGTAAG AATGTTTATA 161ATAACAACTG GTTATCCTGG TTTCAGCAGA AACCAGGGCA 201 GCCTCCCAAG CTCCTGATCTATTATGCATC CACTCTGGCA 241 TCTGGGGTCC CATCGCGGTT CAGAGGCAGT GGATCTGGGA281 CACAGTTCAC TCTCACCATT AGCGACGTGC AGTGTGACGA 321 TGCTGCCACTTACTACTGTG CAGGCGATTA TAGTAGTAGT 361 AGTGATAATG GTTTCGGCGG AGGGACCGAGGTGGTGGTCA 401 AAGGTGATCC AGTTGCACCT ACTGTCCTCC TCTTCCCACC 441ATCTAGCGAT GAGGTGGCAA CTGGAACAGT CACCATCGTG 481 TGTGTGGCGA ATAAATACTTTCCCGATGTC ACCGTCACCT 521 GGGAGGTGGA TGGCACCACC CAAACAACTG GCATCGAGAA561 CAGTAAAACA CCGCAGAATT CTGCAGATTG TACCTACAAC 601 CTCAGCAGCACTCTGACACT GACCAGGACA CAGTACAACA 641 GCCACAAAGA GTACACCTGC AAGGTGACCCAGGGCACGAC 681 CTCAGTCGTC CAGAGCTTCA GTAGGAAGAA CTGTTAA

In another embodiment, the invention provides a 11G05 heavy chain thatcan bind to CD83 polypeptides. The amino acid sequence for this 11G05heavy chain is provided below (SEQ ID NO:17).

  1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC  41 TVSGFTISDYDLSWVRQAPG EGLKYIGFIA IDGNPYYATW  81 AKGRFTISKT STTVDLKITA PTTEDTATYFCARGAGDLWG 121 PGTLVTVSSG QPKAPSVFPL APCCGDTPSS TVTLGCLVKG 161YLPEPVTVTW NSGTLTNGVR TFPSVRQSSG LYSLSSVVSV 201 TSSSQPVTCN VAHPATNTKVDKTVAPSTCS KPTCPPPELL 241 GGPSVFIFPP KPKDTLMISR TPEVTCVVVD VSQDDPEVQF281 TWYINNEQVR TARPPLREQQ FNSTTRVVST LPIAHQDWLR 321 GKEFKCKVHNKALPAPIEKT ISKARGQPLE PKVYTMGPPR 361 EELSSRSVSL TCMINGFYPS DISVEWEKNGKAEDNYKTTP 401 AVLDSDGSYF LYNKLSVPTS EWQRGDVFTC SVMHEALHNH 441YTQKSISRSP GK

A nucleic acid sequence for this 11G05 anti-CD83 heavy chain is providedbelow (SEQ ID NO:18).

   1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC   41 TCAAAGGTGTCCAGTGTCAG TCGGTGGAGG AGTCCGGGGG   81 TCGCCTGGTC ACGCCTGGGA CACCCCTGACACTCACCTGC  121 ACAGTCTCTG GATTCACCAT CAGTGACTAC GACTTGAGCT  161GGGTCCGCCA GGCTCCAGGG GAGGGGCTGA AATACATCGG  201 ATTCATTGCT ATTGATGGTAACCCATACTA CGCGACCTGG  241 GCAAAAGGCC GATTCACCAT CTCCAAAACC TCGACCACGG 281 TGGATCTGAA AATCACCGCT CCGACAACCG AAGACACGGC  321 CACGTATTTCTGTGCCAGAG GGGCAGGGGA CCTCTGGGGC  361 CCAGGGACCC TCGTCACCGT CTCTTCAGGGCAACCTAAGG  401 CTCCATCAGT CTTCCCACTG GCCCCCTGCT GCGGGGACAC  441ACCCTCTAGC ACGGTGACCT TGGGCTGCCT GGTCAAAGGC  481 TACCTCCCGG AGCCAGTGACCGTGACCTGG AACTCGGGCA  521 CCCTCACCAA TGGGGTACGC ACCTTCCCGT CCGTCCGGCA 561 GTCCTCAGGC CTCTACTCGC TGAGCAGCGT GGTGAGCGTG  601 ACCTCAAGCAGCCAGCCCGT CACCTGCAAC GTGGCCCACC  641 CAGCCACCAA CACCAAAGTG GACAAGACCGTTGCGCCCTC  681 GACATGCAGC AAGCCCACGT GCCCACCCCC TGAACTCCTG  721GGGGGACCGT CTGTCTTCAT CTTCCCCCCA AAACCCAAGG  761 ACACCCTCAT GATCTCACGCACCCCCGAGG TCACATGCGT  801 GGTGGTGGAC GTGAGCCAGG ATGACCCCGA GGTGCAGTTC 841 ACATGGTACA TAAACAACGA GCAGGTGCGC ACCGCCCGGC  881 CGCCGCTACGGGAGCAGCAG TTCAACAGCA CGATCCGCGT  921 GGTCAGCACC CTCCCCATCG CGCACCAGGACTGGCTGAGG  961 GGCAAGGAGT TCAAGTGCAA AGTCCACAAC AAGGCACTCC 1001CGGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAGAGGGCA 1041 GCCCCTGGAG CCGAAGGTCTACACCATGGG CCCTCCCCGG 1081 GAGGAGCTGA GCAGCAGGTC GGTCAGCCTG ACCTGCATGA1120 TCAACGGCTT CTACCCTTCC GACATCTCGG TGGAGTGGGA 1161 GAAGAACGGGAAGGCAGAGG ACAACTACAA GACCACGCCG 1201 GCCGTGCTGG ACAGCGACGG CTCCTACTTCCTCTACAACA 1241 AGCTCTCAGT GCCCACGAGT GAGTGGCAGC GGGGCGACGT 1281CTTCACCTGC TCCGTGATGC ACGAGGCCTT GCACAACCAC 1321 TACACGCAGA AGTCCATCTCCCGCTCTCCG GGTAAA

In another embodiment, the invention provides a 14C12 light chain thatcan bind to CD83 polypeptides. The amino acid sequence for this 14C12light chain is provided below (SEQ ID NO:19).

  1 MDXRAPTQLL GLLLLWLPGA RCALVMTQTP ASVSAAVGGT  41 VTINCQSSQSVYDNDELSWY QQKPGQPPKL LIYLASKLAS  81 GVPSRFKGSG SGTQFALTIS GVQCDDAATYYCQATHYSSD 121 WYLTFGGGTE VVVKGDPVAP TVLLFPPSSD EVATGTVTIV 161CVANKYFPDV TVTWEVDGTT QTTGIENSKT PQNSADCTYH 201 LSSTLTLTST QYNSHKEYTCKVTQGTTSVV QSFSRKNC

A nucleic acid sequence for this 14C12 anti-CD83 light chain is providedbelow (SEQ ID NO:20).

  1 ATGGACATRA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC  41 TGCTCTGGCTCCCAGGTGCC AGATGTGCCC TTGTGATGAC  81 CCAGACTCCA GCCTCCGTGT CTGCAGCTGTGGGAGGCACA 121 GTCACCATCA ATTGCCAGTC CAGTCAGAGT GTTTATGATA 161ACGACGAATT ATCCTGGTAT CAGCAGAAAC CAGGGCAGCC 201 TCCCAAGCTC CTGATCTATCTGGCATCCAA GTTGGCATCT 241 GGGGTCCCAT CCCGATTCAA AGGCAGTGGA TCTGGGACAC281 AGTTCGCTCT CACCATCAGC GGCGTGCAGT GTGACGATGC 321 TGCCACTTACTACTGTCAAG CCACTCATTA TAGTAGTGAT 361 TGGTATCTTA CTTTCGGCGG AGGGACCGAGGTGGTGGTCA 401 AAGGTGATCC AGTTGCACCT ACTGTCCTCC TCTTCCCACC 441ATCTAGCGAT GAGGTGGCAA CTGGAACAGT CACCATCGTG 481 TGTGTGGCGA ATAAATACTTTCCCGATGTC ACCGTCACCT 521 GGGAGGTGGA TGGCACCACC CAAACAACTG GCATCGAGAA561 CAGTAAAACA CCGCAGAATT CTGCAGATTG TACCTACAAC 601 CTCAGCAGCACTCTGACACT GACCAGCACA CAGTACAACA 641 GCCACAAAGA GTACACCTGC AAGGTGACCCAGGGCACGAC 681 CTCAGTCGTC CAGAGCTTCA GTAGGAAGAA CTGTTAA

In another embodiment, the invention provides a 14C12 heavy chain thatcan bind to CD83 polypeptide's. The amino acid sequence for this 14C12heavy chain is provided below (SEQ ID NO:21).

  1 METGLRWLLL VAVLKGVHCQ SVEESGGRLV TPGTPLTLTC  41 TASGFSRSSYDMSWVRQAPG KGLEWVGVIS TAYNSHYASW  81 AKGRFTISRT STTVDLKMTS LTTEDTATYFCARGGSWLDL 121 WGQGTLVTVS SGQPKAPSVF PLAPCCGDTP SSTVTLGCLV 161KGYTPEPVTV TWNSGTLTNG VRTFPSVRQS SGLYSLSSVV 201 SVTSSSQPVT CNVAHPATNTKVDKTVAPST CSKPTCPPPE 241 LLGGPSVFIF PPKPKDTLMI SRTPEVTCVV VDVSQDDPEV281 QFTWYINNEQ VRTARPPLRE QQFNSTIRVV STLPIAHQDW 321 LRGKEFKCKVHNKALPAPIE KTISKARGQP LEPKVYTMGP 361 PREELSSRSV SLTCMTNGFY PSDISVEWEKNGKAEDNYKT 401 TPAVLDSDGS YFLYNKLSVP TSEWQRGDVF TCSVMHEALH 441NHYTQKSISR SPGK

A nucleic acid sequence for this 14C12 anti-CD83 heavy chain is providedbelow (SEQ ID NO:22).

   1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC   41 TCAAAGGTGTCCACTGTCAG TCGGTGGAGG AGTCCGGGGG   81 TCGCCTGGTC ACGCCTGGGA CACCCCTGACACTCACCTGC  121 ACAGCCTCTG GATTCTCCCG CAGCAGCTAC GACATGAGCT  161GGGTCCGCCA GGCTCCAGGG AAGGGGCTGG AATGGGTCGG  201 AGTCATTAGT ACTGCTTATAACTCACACTA CGCGAGCTGG  241 GCAAAAGGCC GATTCACCAT CTCCAGAACC TCGACCACGG 281 TGGATCTGAA AATGACCAGT CTGACAACCG AAGACACGGC  321 CACCTATTTCTGTGCCAGAG GGGGTAGTTG GTTGGATCTC  361 TGGGGCCAGG GCACCCTGGT CACCGTCTCCTCAGGGCAAC  401 CTAAGGCTCC ATCAGTCTTC CCACTGGCCC CCTGCTGCGG  441GGACACACCC TCTAGCACGG TGACCTTGGG CTGCCTGGTC  481 AAAGGCTACC TCCCGGAGCCAGTGACCGTG ACCTGGAACT  521 CGGGCACCCT CACCAATGGG GTACGCACCT TCCCGTCCGT 561 CCGGCAGTCC TCAGGCCTCT ACTCGCTGAG CAGCGTGGTG  601 AGCGTGACCTCAAGCAGCCA GCCCGTCACC TGCAACGTGG  641 CCCACCCAGC CACCAACACC AAAGTGGACAAGACCGTTGC  681 GCCCTCGACA TGCAGCAAGC CCACGTGCCC ACCCCCTGAA  721CTCCTGGGG3 GACCGTCTGT CTTCATCTTC CCCCCAAAAC  761 CCAAGGACAC CCTCATGATCTCACGCACCC CCGAGGTCAC  801 ATGCGTGGTG GTGGACGTGA GCCAGGATGA CCCCGAGGTG 841 CAGTTCACAT GGTACATAAA CAACGAGCAG GTGCGCACCG  881 CCCGGCCGCCGCTACGGGAG CAGCAGTTCA ACAGCACGAT  921 CCGCGTGGTC AGCACCCTCC CCATCGCGCACCAGGACTGG  961 CTGAGGGGCA AGGAGTTCAA GTGCAAAGTC CACAACAAGG 1001CACTCCCGGC CCCCATCGAG AAAACCATCT CCAAAGCCAG 1041 AGGGCAGCCC CTGGAGCCGAAGGTCTACAC CATGGGCCCT 1081 CCCCGGGAGG AGCTGAGCAG CAGGTCGGTC AGCCTGACCT1121 GCATGATCAA CGGCTTCTAC CCTTCCGACA TCTCGGTGGA 1161 GTGGGAGAAGAACGGGAAGG CAGAGGACAA CTACAAGACC 1200 ACGCCGGCCG TGCTGGACAG CGACGGCTCCTACTTCCTCT 1241 ACAACAAGCT CTCAGTGCCC ACGAGTGAGT GGCAGCGGGG 1281CGACGTCTTC ACCTGCTCCG TGATGCACGA GGCCTTGCAC 1321 AACCACTACA CGCAGAAGTCCATCTCCCGC TCTCCGGGTA 1361 AA

In another embodiment, the invention provides a M83 020B08L light chainthat can bind to CD83 polypeptides. The amino acid sequence for this M83020B08L light chain is provided below (SEQ ID NO:58).

  1 MDMRAPTQLL GLLLLWLPGA RCAYDMTQTP ASVEVAVGGT  41 VTIKCQASQSISTYLDWYQQ KPGQPPKLLI YDASDIASGV  81 PSRFKGSGSG TQFTLTISDL ECADAATYYCQQGYTHSNVD 121 NVFGGGTEVV VKGDPVAPTV LLFPPSSDEV ATGTVTIVCV 161ANKYFPDVTV TWEVDGTTQT TGIENSKTPQ NSADCTYNLS 201 STLTLTSTQY NSHKEYTCKVTQGTTSVVQS FSRKNC

A nucleic acid sequence for this M83 020B08L anti-CD83 light chain isprovided below (SEQ ID NO:59).

  1 ATGGACATGA GGGCCCCCAC TCAGCTGCTG GGGCTCCTGC  41 TGCTCTGGCTCCCAGGTGCC AGATGTGCCT ATGATATGAC  81 CCAGACTCCA GCCTCTGTGG AGGTAGCTGTGGGAGGCACA 121 GTCACCATCA AGTGCCAGGC CAGTCAGAGC ATTAGTACCT 161ACTTAGACTG GTATCAGCAG AAACCAGGGC AGCCTCCCAA 201 GCTCCTGATC TATGATGCATCCGATCTGGC ATCTGGGGTC 241 CCATCGCGGT TCAAAGGCAG TGGATCTGGG ACACAGTTCA281 CTCTCACCAT CAGCGACCTG GAGTGTGCCG ATGCTGCCAC 321 TTACTACTGTCAACAGGGTT ATACACATAG TAATGTTGAT 361 AATGTTTTCG GCGGAGGGAC CGAGGTGGTGGTCAAAGGTG 401 ATCCAGTTGC ACCTACTGTC CTCCTCTTCC CACCATCTAG 441CGATGAGGTG GCAACTGGAA CAGTCACCAT CGTGTGTGTG 481 GCGAATAAAT ACTTTCCCGATGTCACCGTC ACCTGGGAGG 521 TGGATGGCAC CACCCAAACA ACTGGCATCG AGAACAGTAA561 AACACCGCAG AATTCTGCAG ATTGTACCTA CAACCTCAGC 601 AGCACTCTGACACTGACCAG CACACAGTAC AACAGCCACA 641 AAGAGTACAC CTGCAAGGTG ACCCAGGGCACGACCTCAGT 681 CGTCCAGAGC TTCAGTAGGA AGAACTGTTA A

In another embodiment, the invention provides a M83 020B08H heavy chainthat can bind to CD83 polypeptides. The amino acid sequence for this M83020B08H heavy chain is provided below (SEQ ID NO:60).

  1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV TPGTPLTLTC  41 TVSGFSLSSYDMTNVRQAPG KGLEWIGIIY ASGTTYYANW  81 AKGRFTISKT STTVDLKVTS PTIGDTATYFCAREGAGVSM 121 TLWGPQTLVT VSSGQPKAPS VFPLAPCCGD TPSSTVTLGC 161LVKGYLPEPV TVTWNSGTLT NGVRTFPSVR QSSGLYSLSS 201 VVSVTSSSQP VTCNVAHPATNTKVDKTVAP STCSKPTCPP 241 PELLGGPSVF IFPPKPKDTL MISRTPEVTC VVVDVSQDDP281 EVQFTWYINN EQVRTARPPL REQQFNSTIR VVSTLPIAHQ 321 DWLRGKEFKCKVHNKALPAP IEKTISKARG QPLEPKVYTM 361 GPPREELSSR SVSLTCMING FYPSDISVEWEKNGKAEDNY 401 KTTPAVLDSD GSYFLYNKLS VPTSEWQRGD VFTCSVMHEA 441LHNHYTQKSI SRSPGK

A nucleic acid sequence for this M83 020B08H anti-CD83 heavy chain isprovided below (SEQ ID NO:61).

   1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC   41 TCAAAGGTGTCCAGTGTCAG TCGGTGGAGG AGTCCGGGGG   81 TCGCCTGGTC ACGCCTGGGA CACCCCTGACACTCACCTGC  121 ACAGTCTCTG GATTCTCCCT CAGCAGCTAC GACATGACCT  161GGGTCCGCCA GGCTCCAGGG AAGGGGCTGG AATGGATCGG  201 AATCATTTAT GCTAGTGGTACCACATACTA CGCGAACTGG  241 GCGAAAGGCC GATTGACCAT CTCCAAAACC TCGACCACGG 281 TGGATCTGAA AGTCACCAGT CCGACAATCG GGGACACGGC  321 CACCTATTTCTGTGCCAGAG AGGGGGCTGG TGTTAGTATG  361 ACCTTGTGGG GCCCAGGCAC CCTGGTCACCGTCTCCTCAG  401 GGCAACCTAA GGCTCCATCA GTCTTCCCAC TGGCCCCCTG  441CTGCGGGGAC ACACCCTCTA GCACGGTGAC CTTGGGCTGC  481 CTGGTCAAAG GCTACCTCCCGGAGCCAGTG ACCGTGACCT  521 GGAACTCGGG CACCCTCACC AATGGGGTAC GCACCTTCCC 561 GTCCGTCCGG CAGTCCTCAG GCCTCTACTC GCTGAGCAGC  601 GTGGTGAGCGTGACCTCAAG CAGCCAGCCC GTCACCTGCA  641 ACGTGGCCCA CCCAGCCACC AACACCAAAGTGGACAAGAC  681 CGTTGCGCCC TCGACATGCA GCAAGCCCAC GTGCCCACCC  721CCTGAACTCC TGGGGGGACC GTCTGTCTTC ATCTTCCCCC  761 CAAAACCCAA GGACACCCTCATGATCTCAC GCACCCCCGA  801 GGTCACATGC GTGGTGGTGG ACGTGAGCCA GGATGACCCC 841 GAGGTGCAGT TCACATGGTA CATAAACAAC GAGCAGGTGC  881 GCACCGCCCGGCCGCCGCTA CGGGAGCAGC AGTTCAACAG  921 CACGATCCGC GTGGTCAGCA CCCTCCCCATCGCGCACCAG  961 GACTGGCTGA GGGGCAAGGA GTTCAAGTGC AAAGTCCACA 1001ACAAGGCACT CCCGGCCCCC ATCGAGAAAA CCATCTCCAA 1041 AGCCAGAGGG CAGCCCCTGGAGCCGAAGGT CTACACCATG 1081 GGCCCTCCCC GGGAGGAGCT GAGCAGCAGG TCGGTCAGCC1121 TGACCTGCAT GATCAACGGC TTCTACCCTT CCGACATCTC 1161 GGTGGAGTGGGAGAAGAACG GGAAGGCAGA GGACAACTAC 1201 AAGACCACGC CGGCCGTGCT GGACAGCGACGGCTCCTACT 1241 TCCTCTACAA CAAGCTCTCA GTGCCCACGA GTGAGTGGCA 1281GCGGGGCGAC GTCTTCACCT GCTCCGTGAT GCACGAGGCC 1321 TTGCACAACC ACTACACGCAGAAGTCCATC TCCCGCTCTC 1361 CGGGTAAA

In another embodiment, the invention provides a M83 006G05L light chainthat can bind to CD83 polypeptides. The amino acid sequence for this M83006G05L light chain is provided below (SEQ ID NO:62).

  1 MDMRAPTQLL GLLLLWLPGA RCAYDMTQTP ASVEVAVGGT  41 VAIKCQASQSVSSYLAWYQQ KPGQPPKPLI YEASMLAAGV  81 SSRFKGSGSG TDFTLTISDL ECDDAATYYCQQGYSISDID 121 NAFGGGTEVV VKGDPVAPTV LLFPPSSDEV ATGTVTIVCV 161ANKYFPDVTV TWEVDGTTQT TGIENSKTPQ NSADCTYNLS 201 STLTLTSTQY NSHKEYTCKVTQGTTSVVQS FSRKNC

A nucleic acid sequence for M83 006G05L anti-CD83 light chain isprovided below (SEQ ID NO:63).

  1 ATGGACATGA GGGCCCCCAC TCAACTGCTG GGGCTCCTGC  41 TGCTCTGGCTCCCAGGTGCC AGATGTGCCT ATGATATGAC  81 CCAGACTCCA GCCTCTGTGG AGGTAGCTGTGGGAGGCACA 121 GTCGCCATCA AGTGCCAGGC CAGTCAGAGC GTTAGTAGTT 161ACTTAGCCTG GTATCAGCAG AAACCAGGGC AGCCTCCCAA 201 GCCCCTGATC TACGAAGCATCCATGCTGGC GGCTGGGGTC 241 TCATCGCGGT TCAAAGGCAG TGGATCTGGG ACAGACTTCA281 CTCTCACCAT CAGCGACCTG GAGTGTGACG ATGCTGCCAC 321 TTACTATTGTCAACAGGGTT ATTCTATCAG TGATATTGAT 361 AATGCTTTCG GCGGAGGGAC CGAGGTGGTGGTCAAAGGTG 401 ATCCAGTTGC ACCTACTGTC CTCCTCTTCC CACCATCTAG 441CGATGAGGTG GCAACTGGAA CAGTCACCAT CGTGTGTGTG 481 GCGAATAAAT ACTTTCCCGATGTCACCGTC ACCTGGGAGG 521 TGGATGGCAC CACCCAAACA ACTGGCATCG AGAACAGTAA561 AACACCGCAG AATTCTGCAG ATTGTACCTA CAACCTCAGC 601 AGCACTCTGACACTGACCAG CACACAGTAC AACAGCCACA 641 AAGAGTACAC CTGCAAGGTG ACCCAGGGCACGACCTCAGT 681 CGTCCAGAGC TTCAGTAGGA AGAACTGTTA A

In another embodiment, the invention provides a M83 006G05L heavy chainthat can bind to CD83 polypeptides. The amino acid sequence for this M83006G05L heavy chain is provided below (SEQ ID NO:64).

  1 METGLRWLLL VAVLKGVQCQ SVEESGGRLV SPGTPLTLTC  41 TASGFSLSSYDMSWVRQAPG KGLEYIGTIS SSGSTYYASW  81 AKGRFTISKT STTVDLEVTS LTTEDTATYFCSREHAGYSG 121 DTGHLWGPGT LVTVSSGQPK APSVFPLAPC CGDTPSSTVT 161LGCLVKGYLP EPVTVTWNSG TLTNGVRTFP SVRQSSGLYS 201 LSSVVSVTSS SQPVTCNVAHPATNTKVDKT VAPSTCSKPT 241 CPPPELLGGP SVFIFPPKPK DTLMISRTPE VTCVVVDVSQ281 DDPEVQFTWY INNEQVRTAR PPLREQQFNS TIRVVSTLPI 321 AHQDWLRGKEFKCKVHNKAL PAPIEKTISK ARGQPLEPKV 361 YTMGPPREEL SSRSVSLTCM INGPYPSDISVEWEKNGKAE 401 DNYKTTPAVL DSDGSYPLYN KLSVPTSEWQ RGDVFTCSVM 441IIEALHNHYTQ KSISRSPGK

A nucleic acid sequence for this M83 006G05L anti-CD83 heavy chain isprovided below (SEQ ID NO:65).

   1 ATGGAGACAG GCCTGCGCTG GCTTCTCCTG GTCGCTGTGC   41 TCAAAGGTGTCCAGTGTCAG TCGGTGGAGG AGTCCGGGGG   81 TCGCCTGGTC TCGCCTGGGA CACCCCTGACACTCACCTGC  121 ACAGCCTCTG GATTCTCCCT CAGTAGCTAC GACATGAGCT  161GGGTCCGCCA GGCTCCAGGG AAGGGGCTGG AATACATCGG  201 AATCATTAGT AGTAGTGGTAGCACATACTA CGCGAGCTGG  241 GCGAAAGGCC GATTCACCAT CTCCAAAACC TCGACCACGG 281 TGGATCTGGA AGTGACCAGT CTGACAACCG AGGACACGGC  321 CACCTATTTCTGTAGTAGAG AACATGCTGG TTATAGTGGT  361 GATACGGGTC ACTTGTGGGG CCCAGGCACCCTGGTCACCG  401 TCTCCTCGGG GCAACCTAAG GCTCCATCAG TCTTCCCACT  441GGCCCCCTGC TGCGGGGACA CACCCTCTAG CACGGTGACC  481 TTGGGCTGCC TGGTCAAAGGCTACCTCCCG GAGCCAGTGA  521 CCGTGACCTG GAACTCGGGC ACCCTCACCA ATGGGGTACG 561 CACCTTCCCG TCCGTCCGGC AGTCCTCAGG CCTCTACTCG  601 CTGAGCAGCGTGGTGAGCGT GACCTCAAGC AGCCAGCCCG  641 TCACCTGCAA CGTGGCCCAC CCAGCCACCAACACCAAAGT  681 GGACAAGACC GTTGCGCCCT CGACATGCAG CAAGCCCACG  721TGCCCACCCC CTGAACTCCT GGGGGGACCG TCTGTCTTCA  761 TCTTCCCCCC AAAACCCAAGGACACCCTCA TGATCTCACG  801 CACCCCCGAG GTCACATGCG TGGTGGTGGA CGTGAGCCAG 841 GATGACCCCG AGGTGCAGTT CACATGGTAC ATAAACAACG  881 AGCAGGTGCGCACCGCCCGG CCGCCGCTAC GGGAGCAGCA  921 GTTCAACAGC ACGATCCGCG TGGTCAGCACCCTCCCCATC  961 GCGCACCAGG ACTGGCTGAG GGGCAAGGAG TTCAAGTGCA 1001AAGTCCACAA CAAGGCACTC CCGGCCCCCA TCGAGAAAAC 1041 CATCTCCAAA GCCAGAGGGCAGCCCCTGGA GCCGAAGGTC 1081 TACACCATGG GCCCTCCCCG GGAGGAGCTG AGCAGCAGGT1121 CGGTCAGCCT GACCTGCATG ATCAACGGCT TCTACCCTTC 1162 CGACATCTCGGTGGAGTGGG AGAAGAACGG GAAGGCAGAG 1201 GACAACTACA AGACCACGCC GGCCGTGCTGGACAGCGACG 1241 GCTCCTACTT CCTCTACAAC AAGCTCTCAG TGCCCACGAG 1281TGAGTGGCAG CGGGGCGACG TCTTCACCTG CTCCGTGATG 1321 CACGAGGCCT TGGACAACCACTACACGCAG AAGTCCATCT 1361 CCCGCTCTCC GGGTAAA

Anti-Sense Nucleic Acids

Anti-sense nucleic acids can be used to inhibit the function of CD83. Ingeneral, the function of CD83 RNA is inhibited, for example, byadministering to a mammal a nucleic acid that can inhibit thefunctioning of CD83 RNA. Nucleic acids that can inhibit the function ofa CD83RNA can be generated from coding and non-coding regions of theCD83 gene. However, nucleic acids that can inhibit the function of aCD83 RNA are often selected to be complementary to CD83 nucleic acidsthat are naturally expressed in the mammalian cell to be treated withthe methods of the invention. In some embodiments, the nucleic acidsthat can inhibit CD83 RNA functions are complementary to CD83 sequencesfound near the 5′ end of the CD83 coding region. For example, nucleicacids that can inhibit the function of a CD83 RNA can be complementaryto the 5′ region of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ IDNO:10.

A nucleic acid that can inhibit the functioning of a CD83 RNA need notbe 100% complementary to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ IDNO:10. Instead, some variability the sequence of the nucleic acid thatcan inhibit the functioning of a CD83 RNA is permitted. For example, anucleic acid that can inhibit the functioning of a CD83 RNA from a humancan be complementary to a nucleic acid encoding either a human or amouse CD83 gene product.

Moreover, nucleic acids that can hybridize under moderately or highlystringent hybridization conditions to a nucleic acid comprising SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:10 are sufficientlycomplementary to inhibit the functioning of a CD83 RNA and can beutilized in the methods of the invention.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization are somewhatsequence dependent, and may differ depending upon the environmentalconditions of the nucleic acid. For example, longer sequences tend tohybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular biology-Hybridization withNucleic Acid Probes, page 1, chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,N.Y. (1993). See also, J. Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, N.Y., pp 9.31-9.58 (1989);J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, N.Y. (3rd ed. 2001).

Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific double-stranded sequence at a defined ionic strengthand pH. For example, under “highly stringent conditions” or “highlystringent hybridization conditions” a nucleic acid will hybridize to itscomplement to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background). By controlling the stringency of thehybridization and/or washing conditions nucleic acids that are 100%complementary can be hybridized. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl Anal. Biochem.138:267-284 (1984):

T_(m)81.5° C.+16.6(log M)+0.41 (% GC)−0.61 (% form)−500/L

where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe.

Very stringent conditions are selected to be equal to the T_(m), for aparticular probe.

Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity canhybridize. Typically, stringent conditions will be those in which thesalt concentration is less than about 1.5 M Na ion, typically about 0.01to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl and0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

The degree of complementarity or sequence identity of hybrids obtainedduring hybridization is typically a function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. The type and length of hybridizing nucleicacids also affects whether hybridization will occur and whether anyhybrids formed will be stable under a given set of hybridization andwash conditions.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids that have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamidewith 1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent conditions is 0.1 5 M NaCl at72° C. for about 15 minutes. An example of stringent wash conditions isa 0.2×SSC wash at 65° C. for 15 minutes (see also, Sambrook, infra).Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example of medium stringency for aduplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15minutes. An example low stringency wash for a duplex of, e.g., more than100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1.0M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3, and the temperature is typically at least about 30° C.

Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g., when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to detect and isolate homologous nucleic acids that aresubstantially identical to reference nucleic acids of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.,more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

In general, T_(m) is reduced by about 1° C. for each 1% of mismatching.Thus, T_(m), hybridization, and/or wash conditions can be adjusted tohybridize to sequences of the desired sequence identity. For example, ifsequences with >90% identity are sought, the T_(m) can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).

If the desired degree of mismatching results in a T_(m) of less than 45°C. (aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part 1, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley—Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor. Laboratory Press, Plainview, N.Y.).Using these references and the teachings herein on the relationshipbetween T_(m), mismatch, and hybridization and wash conditions, those ofordinary skill can generate variants of the present homocysteineS-methyltransferase nucleic acids.

Precise complementarity is therefore not required for successful duplexformation between a nucleic acid that can inhibit a CD83 RNA and thecomplementary coding sequence of a CD83 RNA. Inhibitory nucleic acidmolecules that comprise, for example, 2, 3, 4, or 5 or more stretches ofcontiguous nucleotides that are precisely complementary to a CD83 codingsequence, each separated by a stretch of contiguous nucleotides that arenot complementary to adjacent CD83 coding sequences, can inhibit thefunction of CD83 RNA. In general, each stretch of contiguous nucleotidesis at least 4, 5, 6, 7, or 8 or more nucleotides in length.Non-complementary intervening sequences are preferably 1, 2, 3, or 4nucleotides in length. One skilled in the art can easily use thecalculated melting point of an anti-sense nucleic acid hybridized to asense nucleic acid to determine the degree of mismatching that will betolerated between a particular anti-sense nucleic acid and a particularCD83 RNA.

Nucleic acids that complementary a CD83 RNA can be administered to amammal or to directly to the site of the inappropriate immune systemactivity. Alternatively, nucleic acids that are complementary to a CD83RNA can generated by transcription from an expression cassette that hasbeen administered to a mammal. For example, a complementary RNA can betranscribed from a CD83 nucleic acid that has been inserted into anexpression cassette in the 3′ to 5′ orientation, that is, opposite tothe usual orientation employed to generate sense RNA transcripts. Hence,to generate a complementary RNA that can inhibit the function of anendogenous CD83 RNA, the promoter would be positioned to transcribe froma 3′ site towards the 5′ end of the CD83 coding region.

In some embodiments an RNA that can inhibit the function of anendogenous CD83 RNA is an anti-sense oligonucleotide. The anti-senseoligonucleotide is complementary to at least a portion of the codingsequence of a gene comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 orSEQ ID NO:10. Such anti-sense oligonucleotides are generally at leastsix nucleotides in length, but can be about 8, 12, 15, 20, 25, 30, 35,40, 45, or 50 nucleotides long. Longer oligonucleotides can also beused. CD83 anti-sense oligonucleotides can be provided in a DNAconstruct and introduced into cells whose division is to be decreased,for example, into CD4+ T cells, Th-1 cells, Th-2 cells or lymphocyteprecursor cells.

Anti-sense oligonucleotides can be composed of deoxyribonucleotides,ribonucleotides, or a combination of both. Oligonucleotides can besynthesized endogenously from transgenic expression cassettes or vectorsas described herein. Alternatively, such oligonucleotides can besynthesized manually or by an automated synthesizer, by covalentlylinking the 5′ end of one nucleotide with the 3′ end of anothernucleotide with non-phosphodiester internucleotide linkages suchalkylphosphonates, phosphorothioates, phosphorodithioates,alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters. See Brown, 1994, Meth. Mol. Biol. 20:1-8; Sonveaux,1994, Meth. Mol. Biol. 26:1-72; Uhlmann et al., 1990, Chem. Rev.90:543-583.

CD83 anti-sense oligonucleotides can be modified without affecting theirability to hybridize to a CD83 RNA. These modifications can be internalor at one or both ends of the anti-sense molecule. For example,internucleoside phosphate linkages can be modified by adding peptidyl,cholesteryl or diamine moieties with varying numbers of carbon residuesbetween these moities and the terminal ribose. Modified bases and/orsugars, such as arabinose instead of ribose, or a 3′,5′-substitutedoligonucleotide in which the 3′ hydroxyl group or the 5′ phosphate groupare substituted, can also be employed in a modified anti-senseoligonucleotide. These modified oligonucleotides can be prepared bymethods available in the art. Agrawal et al., 1992, Trends Biotechnol.10:152-158; Uhlmann et al., 1990, Chem. Rev. 90:543-584; Uhlmann et al.,1987, Tetrahedron. Lett. 215:3539-3542.

In one embodiment of the invention, expression of a CD83 gene isdecreased using a ribozyme. A ribozyme is an RNA molecule with catalyticactivity. See, e.g., Cech, 1987, Science 236: 1532-1539; Cech, 1990,Ann. Rev. Biochem. 59:543-568; Cech, 1992, Curr. Opin. Struct. Biol. 2:605-609; Couture and Stinchcomb, 1996, Trends Genet. 12: 510-515.Ribozymes can be used to inhibit gene function by cleaving an RNAsequence, as is known in the art (see, e.g., Haseloff et al., U.S. Pat.No. 5,641,673).

CD83 nucleic acids complementary to SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5 or SEQ ID NO:10 can be used to generate ribozymes that willspecifically bind to mRNA transcribed from a CD83 gene. Methods ofdesigning and constructing ribozymes that can cleave other RNA moleculesin trans in a highly sequence specific manner have been developed anddescribed in the art (see Haseloff et al. (1988), Nature 334:585-591).For example, the cleavage activity of ribozymes can be targeted tospecific RNAs by engineering a discrete “hybridization” region into theribozyme. The hybridization region contains a sequence complementary tothe target RNA and thus specifically hybridizes with the target (see,for example, Gerlach et al., EP 321,201). The target sequence can be asegment of about 10, 12, 15, 20, or 50 contiguous nucleotides selectedfrom a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5 or SEQ ID NO:10. Longer complementary sequences can be used toincrease the affinity of the hybridization sequence for the target. Thehybridizing and cleavage regions of the ribozyme can be integrallyrelated; thus, upon hybridizing to the target RNA through thecomplementary regions, the catalytic region of the ribozyme can cleavethe target.

Other CD83 Modulating Molecules

A wide variety of molecules may be used to modulate CD83 activity orfunction. Such molecules can also be used to modulate the immune systemindependent of CD83. Compositions and methods for modulating CD83activity or expression can include these molecules as well as othercomponents. Representative examples that are discussed in more detailbelow include transcription factors, RNA-binding factors, organicmolecules, or peptides.

RNA-Binding Factors:

One class of molecules that can be used to modulate cytokine levels orGM-CSF levels by way of the CD83 gene is the RNA binding factors. Suchfactors include those described in PCT/EP01/14820 and other sources.

For example, the HuR protein (Genbank accession number U38175) has theability to specifically bind to CD83 RNA at AU-rich elements or sites.Such AU-rich elements comprise sequences such as AUUUA (SEQ ID NO:49),AUUUUA (SEQ ID NO:50) and AUUUUUA (SEQ ID NO:51). Binding by such HuRproteins to CD83 mRNA is thought to increase the stability, transportand translation of CD83 mRNA, and thereby increase the expression ofCD83 polypeptides. Hence, CD83 expression may be increase byadministering HuR proteins or nucleic acids to a mammal.

Conversely, CD83 expression may be decreased by administering factorsthat block HuR binding to CD83 mRNA. Factors that block HuR bindinginclude proteins or nucleic acids that can bind to the AU-rich elementsnormally bound by HuR, for example, nucleic acids or anti-sense nucleicacids that are complementary to AU-rich elements.

Organic Molecules:

Numerous organic molecules may be used to modulate the immune system.These compounds include any compound that can interact with a componentof the immune system. Such compounds may interact directly with CD83,indirectly with CD83 or with some other polypeptide, cell or factor thatplays a role in the function of the immune system. In some embodiments,the organic molecule can bind to a CD83 polypeptide or a CD83 nucleicacid.

Organic molecules can be tested or assayed for their ability to modulateCD83 activity, CD83 function or for their ability to modulate componentsof the immune system. For example, within one embodiment of theinvention suitable organic molecules may be selected either from achemical library, wherein chemicals are assayed individually, or fromcombinatorial chemical libraries where multiple compounds are assayed atonce, then deconvoluted to determine and isolate the most activecompounds.

Representative examples of such combinatorial chemical libraries includethose described by Agrafiotis et al., “System and method ofautomatically generating chemical compounds with desired properties,”U.S. Pat. No. 5,463,564; Armstrong, R. W., “Synthesis of combinatorialarrays of organic compounds through the use of multiple componentcombinatorial array syntheses,” WO 95/02566; Baldwin, J. J. et al.,“Sulfonamide derivatives and their use,” WO 95/24186; Baldwin, J. J. etal., “Combinatorial dihydrobenzopyran library,” WO 95/30642; Brenner, S“New kit for preparing combinatorial libraries,” WO 95/16918; Chenera,B. et al., “Preparation of library of resin-bound aromatic carbocycliccompounds,” WO 95/16712; Ellman, J. A., “Solid phase and combinatorialsynthesis of benzodiazepine compounds on a solid support,” U.S. Pat. No.5,288,514; Felder, E. et al., “Novel combinatorial compound libraries,”WO 95/16209; Lerner, R. et al., “Encoded combinatorial chemicallibraries,” WO 93/20242; Pavia, M. R. et al., “A method for preparingand selecting pharmaceutically useful non-peptide compounds from astructurally diverse universal library,” WO 95/04277; Summerton, J. E.and D. D. Weller, “Morpholino-subunit combinatorial library and method,”U.S. Pat. No. 5,506,337; Holmes, C., “Methods for the Solid PhaseSynthesis of Thiazolidinones, Metathiazanones, and Derivatives thereof,”WO 96/00148; Phillips, G. B. and G. P. Wei, “Solid-phase Synthesis ofBenzimidazoles,” Tet. Letters 37:4887-90, 1996; Ruhland, B. et al.,“Solid-supported Combinatorial Synthesis of StructurallyDiverse□□-Lactams,” J. Amer. Chem. Soc. 111:253-4, 1996; Look, G. C. etal., “The Identification of Cyclooxygenase-1 Inhibitors from4-Thiazolidinone Combinatorial Libraries,” Bioorg and Med. Chem. Letters6:707-12, 1996.

Peptides:

Peptide molecules that modulate the immune system may be obtainedthrough the screening of combinatorial peptide libraries. Such librariesmay either be prepared by one of skill in the art (see e.g., U.S. Pat.Nos. 4,528,266 and 4,359,535, and Patent Cooperation Treaty PublicationNos. WO 92/15679, WO 92/15677, WO 90/07862, WO 90/02809, or purchasedfrom commercially available sources (e.g., New England Biolabs Ph.D.™Phage Display Peptide Library Kit).

Methods of Using the CD83 Mutant Mouse

In one embodiment, the invention provides a method for identifyingligands, receptors, therapeutic drugs and other molecules that canmodulate the phenotype of the mutant CD83 in vivo. This method involvesadministering a test compound to the mutant CD83 mouse of the inventionand observing whether the compound causes a change in the phenotype ofthe mutant mouse. Changes in phenotype that are of interest includeincreases or decreases in T cells (especially CD4+ T cells), increasesor decreases in GMCSF, IL-2, IL-4 or IL-10 cytokine production,increases or decreases in inflammation, increases or decreases indendritic cell function and other T cell responses known to one of skillin the art.

Test compounds can be screened in vitro to ascertain whether theyinteract directly with CD83. In vitro screening can, for example,identify whether a test compound or molecule can bind to the cytoplasmictail or the membrane-associated portions of CD83. Such information,combined with observation of the in vivo phenotype before and afteradministration of the test compound provides further insight into thefunction of CD83 and provides targets for manipulation T cell activationand other functions modulated by CD83.

The invention is not limited to identification of molecules thatdirectly associate with CD83. The in vivo screening methods providedherein can, also identify test compounds that have an indirect effect onCD83, or that partially or completely replace a function of CD83.

Increases or decreases in T cell numbers can be observed in bloodsamples or in samples obtained from thymus, spleen or lymph nodetissues. In order to observe the activation of T cells and/or theinteraction of T cells and dendritic cells, dendritic cells can bepulsed with antigens ex vivo and then injected into mice to prime CD4+ Tcells in draining lymphoid organs. See Inaba et al., J. Exp. Med. 172:631-640, 1990; Liu, et al., J. Exp. Med. 177: 1299-1307, 1993; Sornasseet al., J. Exp. Med. 175: 15-21, 1992. Antigens can also be depositedintramuscularly and dendritic cells from the corresponding afferentlymphatics can carry that antigen in a form stimulatory for T cells.Bujdoso et al., J. Exp. Med. 170: 1285-1302, 1989. According to theinvention, factors stimulating the interaction of dendritic cells with Tcells in vivo can be identified by administering antigens in this mannerand then observing how T cell respond, e.g. by observing whether T cellactivation occurs.

Increases or decreases in cytokine levels can be observed by methodsprovided herein or by other methods available in the art.

Compositions

The CD83 polypeptides and antibodies of the invention, including theirsalts, are administered so as to achieve a reduction in at least onesymptom associated with an infection, indication or disease.

To achieve the desired effect(s), the polypeptide or antibody, a variantthereof or a combination thereof, may be administered as single ordivided dosages, for example, of at least about 0.01 mg/kg to about 500to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, atleast about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1mg/kg to about 50 to 100 mg/kg of body weight, although other dosagesmay provide beneficial results. The amount administered will varydepending on various factors including, but not limited to, thepolypeptide or antibody chosen, the disease, the weight, the physicalcondition, the health, the age of the mammal, whether prevention ortreatment is to be achieved, and if the polypeptide or antibody ischemically modified. Such factors can be readily determined by theclinician employing animal models or other test systems that areavailable in the art.

Administration of the therapeutic agents in accordance with the presentinvention may be in a single dose, in multiple doses, in a continuous orintermittent manner, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of the CD83 polypeptides andantibodies of the invention may be essentially continuous over apreselected period of time or may be in a series of spaced doses. Bothlocal and systemic administration is contemplated.

To prepare the composition, CD83 polypeptides and antibodies aresynthesized or otherwise obtained, purified as necessary or desired andthen lyophilized and stabilized. The polypeptide or antibody can then beadjusted to the appropriate concentration, and optionally combined withother agents. The absolute weight of a given polypeptide or antibodyincluded in a unit dose can vary widely. For example, about 0.01 toabout 2 g, or about 0.1 to about 500 mg, of at least one polypeptide orantibody of the invention, or a plurality of CD83 polypeptides andantibodies specific for a particular cell type can be administered.Alternatively, the unit dosage can vary from about 0.01 g to about 50 g,from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, fromabout 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5g to about 4 g, or from about 0.5 g to about 2 g.

Daily doses of the CD83 polypeptides or antibodies of the invention canvary as well. Such daily doses can range, for example, from about 0.1g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, fromabout 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/dayto about 2 g/day.

Thus, one or more suitable unit dosage forms comprising the therapeuticCD83 polypeptides or antibodies of the invention can be administered bya variety of routes including oral, parenteral (including subcutaneous,intravenous, intramuscular and intraperitoneal), rectal, dermal,transdermal, intrathoracic, intrapulmonary and intranasal (respiratory)routes. The therapeutic CD83 polypeptides or antibodies may also beformulated for sustained release (for example, using microencapsulation,see WO 94/07529, and U.S. Pat. No. 4,962,091). The formulations may,where appropriate, be conveniently presented in discrete unit dosageforms and may be prepared by any of the methods well known to thepharmaceutical arts. Such methods may include the step of mixing thetherapeutic agent with liquid carriers, solid matrices, semi-solidcarriers, finely divided solid carriers or combinations thereof, andthen, if necessary, introducing or shaping the product into the desireddelivery system.

When the therapeutic CD83 polypeptides or antibodies of the inventionare prepared for oral administration, they are generally combined with apharmaceutically acceptable carrier, diluent or excipient to form apharmaceutical formulation, or unit dosage form. For oraladministration, the CD83 polypeptides or antibodies may be present as apowder, a granular formulation, a solution, a suspension, an emulsion orin a natural or synthetic polymer or resin for ingestion of the activeingredients from a chewing gum. The active CD83 polypeptides orantibodies may also be presented as a bolus, electuary or paste. Orallyadministered therapeutic CD83 polypeptides or antibodies of theinvention can also be formulated for sustained release, e.g., the CD83polypeptides or antibodies can be coated, micro-encapsulated, orotherwise placed within a sustained delivery device. The total activeingredients in such formulations comprise from 0.1 to 99.9% by weight ofthe formulation.

By “pharmaceutically acceptable” it is meant a carrier, diluent,excipient, and/or salt that is compatible with the other ingredients ofthe formulation, and not deleterious to the recipient thereof.

Pharmaceutical formulations containing the therapeutic CD83 polypeptidesor antibodies of the invention can be prepared by procedures known inthe art using well-known and readily available ingredients. For example,the polypeptide or antibody can be formulated with common excipients,diluents, or carriers, and formed into tablets, capsules, solutions,suspensions, powders, aerosols and the like. Examples of excipients,diluents, and carriers that are suitable for such formulations includebuffers, as well as fillers and extenders such as starch, cellulose,sugars, mannitol, and silicic derivatives. Binding agents can also beincluded such as carboxymethyl cellulose, hydroxymethylcellulose,hydroxypropyl methylcellulose and other cellulose derivatives,alginates, gelatin, and polyvinyl-pyrrolidone. Moisturizing agents canbe included such as glycerol, disintegrating agents such as calciumcarbonate and sodium bicarbonate. Agents for retarding dissolution canalso be included such as paraffin. Resorption accelerators such asquaternary ammonium compounds can also be included. Surface activeagents such as cetyl alcohol and glycerol monostearate can be included.Adsorptive carriers such as kaolin and bentonite can be added.Lubricants such as talc, calcium and magnesium stearate, and solidpolyethyl glycols can also be included. Preservatives may also be added.The compositions of the invention can also contain thickening agentssuch as cellulose and/or cellulose derivatives. They may also containgums such as xanthan, guar or carbo gum or gum arabic, or alternativelypolyethylene glycols, bentones and montmorillonites, and the like.

For example, tablets or caplets containing the CD83 polypeptides orantibodies of the invention can include buffering agents such as calciumcarbonate, magnesium oxide and magnesium carbonate. Caplets and tabletscan also include inactive ingredients such as cellulose, pregelatinizedstarch, silicon dioxide, hydroxy propyl methyl cellulose, magnesiumstearate, microcrystalline cellulose, starch, talc, titanium dioxide,benzoic acid, citric acid, corn starch, mineral oil, polypropyleneglycol, sodium phosphate, zinc stearate, and the like. Hard or softgelatin capsules containing at least one polypeptide or antibody of theinvention can contain inactive ingredients such as gelatin,microcrystalline cellulose, sodium lauryl sulfate, starch, talc, andtitanium dioxide, and the like, as well as liquid vehicles such aspolyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coatedcaplets or tablets containing one or more CD83 polypeptides orantibodies of the invention are designed to resist disintegration in thestomach and dissolve in the more neutral to alkaline environment of theduodenum.

The therapeutic CD83 polypeptides or antibodies of the invention canalso be formulated as elixirs or solutions for convenient oraladministration or as solutions appropriate for parenteraladministration, for instance by intramuscular, subcutaneous,intraperitoneal or intravenous routes. The pharmaceutical formulationsof the therapeutic CD83 polypeptides or antibodies of the invention canalso take the form of an aqueous or anhydrous solution or dispersion, oralternatively the form of an emulsion or suspension or salve.

Thus, the therapeutic CD83 polypeptides or antibodies may be formulatedfor parenteral administration (e.g., by injection, for example, bolusinjection or continuous infusion) and may be presented in unit dose formin ampules, pre-filled syringes, small volume infusion containers or inmulti-dose containers. As noted above, preservatives can be added tohelp maintain the shelve life of the dosage form. The active CD83polypeptides or antibodies and other ingredients may form suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active CD83 polypeptides or antibodies andother ingredients may be in powder form, obtained by aseptic isolationof sterile solid or by lyophilization from solution, for constitutionwith a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable carriers,vehicles and adjuvants that are well known in the art. It is possible,for example, to prepare solutions using one or more organic solvent(s)that is/are acceptable from the physiological standpoint, chosen, inaddition to water, from solvents such as acetone, ethanol, isopropylalcohol, glycol ethers such as the products sold under the name“Dowanol,” polyglycols and polyethylene glycols, C₁-C₄ alkyl esters ofshort-chain acids, ethyl or isopropyl lactate, fatty acid triglyceridessuch as the products marketed under the name “Miglyol,” isopropylmyristate, animal, mineral and vegetable oils and polysiloxanes.

It is possible to add, if necessary, an adjuvant chosen fromantioxidants, surfactants, other preservatives, film-forming,keratolytic or comedolytic agents, perfumes, flavorings and colorings.Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole,butylated hydroxytoluene and α-tocopherol and its derivatives can beadded.

Also contemplated are combination products that include one or more CD83polypeptides or antibodies of the present invention and one or moreother anti-microbial agents. For example, a variety of antibiotics canbe included in the pharmaceutical compositions of the invention, such asaminoglycosides (e.g., streptomycin, gentamicin, sisomicin, tobramycinand amicacin), ansamycins (e.g. rifamycin), antimycotics (e.g. polyenesand benzofuran derivatives), β-lactams (e.g. penicillins andcephalosporins), chloramphenical (including thiamphenol andazidamphenicol), linosamides (lincomycin, clindamycin), macrolides(erythromycin, oleandomycin, spiramycin), polymyxins, bacitracins,tyrothycin, capreomycin, vancomycin, tetracyclines (includingoxytetracycline, minocycline, doxycycline), phosphomycin and fusidicacid.

Additionally, the CD83 polypeptides or antibodies are well suited to toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activepolypeptide or antibody, for example, in a particular part of theintestinal or respiratory tract, possibly over a period of time.Coatings, envelopes, and protective matrices may be made, for example,from polymeric substances, such as polylactide-glycolates, liposomes,microemulsions, microparticles, nanoparticles, or waxes. These coatings,envelopes, and protective matrices are useful to coat indwellingdevices, e.g., stents, catheters, peritoneal dialysis tubing, drainingdevices and the like.

For topical administration, the therapeutic agents may be formulated asis known in the art for direct application to a target area. Formschiefly conditioned for topical application take the form, for example,of creams, milks, gels, dispersion or microemulsions, lotions thickenedto a greater or lesser extent, impregnated pads, ointments or sticks,aerosol formulations (e.g., sprays or foams), soaps, detergents, lotionsor cakes of soap. Other conventional forms for this purpose includewound dressings, coated bandages or other polymer coverings, ointments,creams, lotions, pastes, jellies, sprays, and aerosols. Thus, thetherapeutic CD83 polypeptides or antibodies of the invention can bedelivered via patches or bandages for dermal administration.Alternatively, the polypeptide or antibody can be formulated to be partof an adhesive polymer, such as polyacrylate or acrylate/vinyl acetatecopolymer. For long-term applications it might be desirable to usemicroporous and/or breathable backing laminates, so hydration ormaceration of the skin can be minimized. The backing layer can be anyappropriate thickness that will provide the desired protective andsupport functions. A suitable thickness will generally be from about 10to about 200 microns.

Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. The active CD83 polypeptides or antibodies can also bedelivered via iontophoresis, e.g., as disclosed in U.S. Pat. Nos.4,140,122; 4,383,529; or 4,051,842. The percent by weight of atherapeutic agent of the invention present in a topical formulation willdepend on various factors, but generally will be from 0.01% to 95% ofthe total weight of the formulation, and typically 0.1-85% by weight.

Drops, such as eye drops or nose drops, may be formulated with one ormore of the therapeutic CD83 polypeptides or antibodies in an aqueous ornon-aqueous base also comprising one or more dispersing agents,solubilizing agents or suspending agents. Liquid sprays are convenientlydelivered from pressurized packs. Drops can be delivered via a simpleeye dropper-capped bottle, or via a plastic bottle adapted to deliverliquid contents dropwise, via a specially shaped closure.

The therapeutic polypeptide or antibody may further be formulated fortopical administration in the mouth or throat. For example, the activeingredients may be formulated as a lozenge further comprising a flavoredbase, usually sucrose and acacia or tragacanth; pastilles comprising thecomposition in an inert base such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the composition of the presentinvention in a suitable liquid carrier.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that areavailable in the art. Examples of such substances include normal salinesolutions such as physiologically buffered saline solutions and water.Specific non-limiting examples of the carriers and/or diluents that areuseful in the pharmaceutical formulations of the present inventioninclude water and physiologically acceptable buffered saline solutionssuch as phosphate buffered saline solutions pH 7.0-8.0.

The CD83 polypeptides or antibodies of the invention can also beadministered to the respiratory tract. Thus, the present invention alsoprovides aerosol pharmaceutical formulations and dosage forms for use inthe methods of the invention. In general, such dosage forms comprise anamount of at least one of the agents of the invention effective to treator prevent the clinical symptoms of a specific infection, indication ordisease. Any statistically significant attenuation of one or moresymptoms of an infection, indication or disease that has been treatedpursuant to the method of the present invention is considered to be atreatment of such infection, indication or disease within the scope ofthe invention.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof the therapeutic agent and a suitable powder base such as lactose orstarch. The powder composition may be presented in unit dosage form in,for example, capsules or cartridges, or, e.g., gelatin or blister packsfrom which the powder may be administered with the aid of an inhalator,insufflator, or a metered-dose inhaler (see, for example, thepressurized metered dose inhaler (MDI) and the dry powder inhalerdisclosed in Newinan, S. P. in Aerosols and the Lung, Clarke, S. W. andDavia, D. eds., pp. 197-224, Butterworths, London, England, 1984).

Therapeutic CD83 polypeptides or antibodies of the present invention canalso be administered in an aqueous solution when administered in anaerosol or inhaled form. Thus, other aerosol pharmaceutical formulationsmay comprise, for example, a physiologically acceptable buffered salinesolution containing between about 0.1 mg/ml and about 100 mg/ml of oneor more of the CD83 polypeptides or antibodies of the present inventionspecific for the indication or disease to be treated. Dry aerosol in theform of finely divided solid polypeptide or antibody or nucleic acidparticles that are not dissolved or suspended in a liquid are alsouseful in the practice of the present invention. CD83 polypeptides orantibodies of the present invention may be formulated as dusting powdersand comprise finely divided particles having an average particle size ofbetween about 1 and 5 μm, alternatively between 2 and 3 μm. Finelydivided particles may be prepared by pulverization and screen filtrationusing techniques well known in the art. The particles may beadministered by inhaling a predetermined quantity of the finely dividedmaterial, which can be in the form of a powder. It will be appreciatedthat the unit content of active ingredient or ingredients contained inan individual aerosol dose of each dosage form need not in itselfconstitute an effective amount for treating the particular infection,indication or disease since the necessary effective amount can bereached by administration of a plurality of dosage units. Moreover, theeffective amount may be achieved using less than the dose in the dosageform, either individually, or in a series of administrations.

For administration to the upper (nasal) or lower respiratory tract byinhalation, the therapeutic CD83 polypeptides or antibodies of theinvention are conveniently delivered from a nebulizer or a pressurizedpack or other convenient means of delivering an aerosol spray.Pressurized packs may comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Nebulizers include, butare not limited to, those described in U.S. Pat. Nos. 4,624,251;3,703,173; 3,561,444; and 4,635,627. Aerosol delivery systems of thetype disclosed herein are available from numerous commercial sourcesincluding Fisons Corporation (Bedford, Mass.), Schering Corp.(Kenilworth, N.J.) and American Pharmoseal Co., (Valencia, Calif.). Forintra-nasal administration, the therapeutic agent may also beadministered via nose drops, a liquid spray, such as via a plasticbottle atomizer or metered-dose inhaler. Typical of atomizers are theMistometer (Wintrop) and the Medihaler (Riker).

Furthermore, the active ingredients may also be used in combination withother therapeutic agents, for example, pain relievers, anti-inflammatoryagents, antihistamines, bronchodilators and the like, whether for theconditions described or some other condition.

The present invention further pertains to a packaged pharmaceuticalcomposition for controlling microbial infections such as a kit or othercontainer. The kit or container holds a therapeutically effective amountof a pharmaceutical composition for controlling microbial infections andinstructions for using the pharmaceutical composition for control of themicrobial infection. The pharmaceutical composition includes at leastone polypeptide or antibody of the present invention, in atherapeutically effective amount such that the selected disease orimmunological condition is controlled.

The invention will be further described by reference to the followingdetailed examples, which are given for illustration of the invention,and are not intended to be limiting thereof.

Example 1 Mouse Mutation and Characterization Mutant Generation

Male C57BL6 mice received 3 weekly injections of N-ethyl-N-nitrosourea(ENU) at a concentration of 100 mg/kg. N-Ethyl-N-nitrosourea wasquantified prior to injection by spectrophotometry. Mice that regainedfertility after a minimum period of 12 weeks were then used to generatepedigree founder G1 animals. G1 male mice were crossed to C57BL6Jfemales and their female progeny (G2 animals) crossed back to theirfathers to generate G3 animals for screening.

G3 mice were weaned at 3 weeks of age. Each animal then underwent aseries of screens designed to assess a number of parameters, includingimmune function, inflammatory response and bone development. In theinitial screen, conducted at 6 weeks of age, 150-200 ul of whole bloodwas collected by retro-orbital bleed into heparinized tubes. Cells werepelleted and red blood cells lysed. Samples were then stained withantibodies to cell surface markers expressed on distinct lymphoid andmyeloid sub-populations. These samples were analyzed by flow-cytometry.

Mutant Identification

A group of 27 G3 mice from 2 different pedigrees, pedigree 9 andpedigree 57 (i.e. derived from 2 distinct G1 fathers) were analyzed inthis screen. Two animals from pedigree 9 were identified as having areduced (>2 standard deviation from normal) percentage of CD4+ T cellsin peripheral blood (FIG. 1). Both animals were descended from the sameG1 and shared the same mother. All other animals screened on that dayhad a normal percentage of CD4+ T cells. The number of phenodeviantsidentified (2 from a litter of 9 animals) was suggestive of a traitcontrolled by a single gene and inherited in a Mendelian fashion.

A second litter generated from Pedigree 9 bred to G2 daughter #4exhibited an identical phenotype with reduced numbers of CD4+ T cells,further suggesting that the trait had a genetic basis. The phenotype wasdesignated LCD4.1 (Low CD4 Mutant 1) and was used for mappingexperiments.

Mutation Mapping

In order to map the LCD4.1 mutant phenotype, affected G3 male mice(presumptive homozygous for the mutation) were bred to female animalsfrom the C3HeB/FeJ strain to generate F1 progeny. These F1 females(presumptively heterozygous for the mutation) were then mated back totheir affected father to generate N2 progeny.

Blood was collected from N2 animals and flow cytometric analysis wasperformed to identify CD4+ T cells. For a phenotype controlled by asingle gene, breeding homozygous fathers to heterozygous daughtersshould yield 50% normal N2 animals and 50% affected N2 animals. Thisratio of normal to affected animals was observed in the N2 generation:Multiple N2 animals exhibited a reduced percentage of CD4+ T cells,indicating that the phenotype was heritable (FIG. 2).

DNA samples were prepared from samples of tail tissue collected fromthese N2 mice and used for a genome scan, using a collection ofassembled markers, and performed on the ABI 3100 DNA analyzer. Initialgenetic linkage was seen to the tip of chromosome 13, where the closestmicrosatellite marker was D13Mit139 with a LOD score of 8.2. Bycalculating upper and lower confidence limits, the mutant gene waslocated between 13.4 and 29.6 cM on chromosome 13. Through additionalgenotyping, this region was reduced to an 11 cM interval on chromosome13. No significant linkage to other chromosomal regions was seen.

Mutation Identification

A candidate gene, CD83, was identified for gene-testing based upon itsreported position within the interval. CD83 has previously been used asa marker of dendritic cell activation, suggesting that it might play arole in dendritic cell function and hence in regulating T celldevelopment and function.

Sequence analysis of the mutant DNA revealed a mutation in the stopcodon of CD83. All affected animals were homozygous for this mutationwhile non-affected animals carried one wild-type allele and one mutantallele (FIG. 3 and FIG. 4). The mutation destroyed the stop codon andresulted in the addition of a unique 55 amino acid tail to theC-terminus of CD83 (FIG. 5).

Additional Functional Data

A reduction in CD4+ T cells was seen in peripheral blood, spleen tissuesand lymph nodes from homozygous LCD4.1 mice. Although there was areduced number of CD4+ T cells in the thymus there is no overt block inthe developmental process and there was no alteration in B celldevelopment in the bone marrow. Histological evaluation of thymus,spleen and lymph nodes from affected mice revealed no gross alterationin tissue architecture.

Dendritic cells can be differentiated from bone marrow of wild type miceby culture in GM-CSF. These cells can be characterized by the surfaceexpression of dendritic cell markers, including CD86 and CD11c. BothLCD4.1 affected and normal animals were capable of giving rise toCD86+CD11+ cells under these culture conditions. LCD4.1 mutant mice thuswere capable of generating dendritic cells under in vitro cultureconditions. These data suggest that the phenotype seen in LCD4.1 mice isnot due to a failure of dendritic cells to develop but rather mayreflect a defect in function.

To track dendritic cells the sensitizing agent FITC was applied to thedorsal surface of the ears of LCD4.1 affected and wild-type mice. FITCwas picked up by dendritic cells that then migrated to the drainingauricular lymph nodes, where the presence of the FITC label on thedendritic cell surface permitted detection by flow-cytometry. FITClabeled cells expressing CD86 were detected in equal proportions indraining lymph node from normal and affected LCD4.1 mice. These dataindicate that LCD4.1 mutant animals are capable of generating dendriticcells in vivo and that these cells are able to pick up antigen in theear and travel to the draining lymph node.

Example 2 CD83 and CD4+ T Cell Function Materials and Methods

Spleens were removed from wild type and mutant mice and digested withcollagenase to liberate dendritic cells. Spleens were stained forsurface expression of CD4 (helper T cells) and CD11c (dendritic cells).Cells expressing these markers were purified by fluorescence activatedcell sorting (FACS sorting). CD11c and CD4+ positive cells were alsopurified from an allogeneic mouse strain, BALBc.

Mixed lymphocyte cultures were set up using purified cell populations.Dendritic cells from BALBc animals were used to stimulate CD4+ T cellsfrom wild type and mutant mice. In a reciprocal experiment dendriticcells prepared from wild type and mutant mice were used to stimulateBALBc CD4+ T cells. After 5 days in culture proliferative responses weremeasured by incorporation of tritiated thymidine.

Dendritic cells from wild type and mutant mice were both capable ofactivating allogeneic T cells, suggesting that dendritic cell functionwas unimpaired in the mutant animal (FIG. 6 a). In contrast CD4+ T cellsfrom mutant animals exhibited a diminished response after 5 days ofstimulation (FIG. 6 b).

These data suggest that the mutation in the CD83 gene has minimal effecton dendritic cells intrinsic function but rather has a profound effectupon T cell activity. The CD4+ T cell therefore may have a novelrequirement for CD83 functionality on T cells during allogeneicactivation. CD83 may be influencing the extent of CD4+ T cell activationor altering the duration of the CD4+ T cell proliferative response. Thetherapeutic manipulation of CD83 may thus represent a mechanism for thespecific regulation of T cell function in the treatment of T cellmediated diseases, including autoimmune disorders. Antibodies capable ofblocking CD83 function may be used as therapeutics in the treatment ofimmune diseases whilst the activation of CD83 may have utility inenhancing immune responses in cancer and other circumstances.

CONCLUSION

Although CD83 has been described as a marker of dendritic cellactivation there is little data as to its function in vivo. The mutationprovided by the invention destabilizes or inactivates the protein andleads to impaired surface expression. As a consequence, CD4+ T cellfunction is impaired although the development of dendritic cells is notinhibited and mutant dendritic cells retain functionality. This resultsin the impaired development of CD4+ T cells. This impaired ability toactivate T cells is also seen in a slight decrease in contactsensitivity responses in LCD4.1 mutant mice.

Example 3 Mutant CD83 have Different Cytokine Levels Than Wild Type Mice

This Example demonstrates that CD4⁺ T-cells from CD83 mutant animalsexpress higher levels of IL-4 and lower levels of IL-2 compared to CD4⁺T-cells from CD83 wild type animals.

Methods for Cell Activation and Cytokine Measurements:

Spleens cells from 6-8-week-old homozygous CD83 wild type or CD83 mutant(LCD4.1) mice were used to isolate CD4⁺ T-cells by positive selectionusing magnetic beads (Miltenyi Biotec). A 96 round bottom plate wascoated with 50 μL, per well of a solution containing either 1 or 10μg/mL of anti-CD3 and 0.1 or 0.2 μg/mL of anti-CD28 antibodies (bothfrom Pharmingen) in PBS overnight. This plate was then washed using 150μL of PBS three times. To this pre-coated plate, 20,000 CD4⁺ T-cells(either wild type or CD83 mutant) were added in a 200 μL final volume ofRPMI containing 10% FBS, 55 μM β-mercaptoethanol and antibiotics. Theplates were then incubated in a CO₂ incubator at 37° C. for 44 to 72hours. For determination of cytokine levels, supernatants were harvestedand cytokines were measured using either a Cytometric Bead Array system(Pharmingen) or ELISA (R&D). For RNA measurements, the cells wereharvested and RNA was isolated using Tri reagent (Sigma). IL-10 and IL-4mRNA levels were measured by reverse transcription and TaqMan (AppliedBiosystems) analysis.

Results:

FIG. 7 shows the IL-2, IL-4, IL-5, TNFα and IFNγ levels produced byeither wild type or CD83 mutant CD4⁺ T-cells. Purified cells wereincubated as described above in the presence of 1 μg/mL of anti-CD3 and0.2 μg/mL of anti-CD28 antibodies for 72 hours. The supernatants werethen simultaneously analyzed for production of IL-2, IL-4, IL-5, TNFαand IFNγ using the cytometric bead array system from Pharmingen.

FIG. 7 demonstrates that CD4⁺ T-cells from CD83 mutant animals expressedhigher levels of IL-4 and lower levels of IL-2 compared to CD4⁺ T-cellsfrom CD83 wild type animals. Other cytokines and a new set ofstimulation assays were analyzed including the production levels ofIL-10 and GMCSF by these cells (FIGS. 8 and 9). In both cases, cellsfrom mutant animals produce larger amounts of IL-10 and GMCSF than didwild type animals. FIG. 10 shows that mRNA levels for both IL-4 andIL-10 were increased in cells from activated mutant CD83, CD4⁺ T-cellscompared with cells from wild type animals.

Example 4 Anti-CD83 Antibodies May Mimic The Effects of the CD83Mutation Methods for Antibody Testing:

For modulation of cytokine production by anti-CD83 antibodies, CD4⁺T-cells were isolated and activated as mentioned above in the presenceof increasing concentrations of anti-CD83 antibodies. For proliferationassays, CD4⁺ T-cells were isolated from an OT2tg [transgenic mice with aT-cell receptor specific for chicken ovalbumin (OVA) 323-339 peptide].Dendritic cells were isolated from a C57BL6 mouse by a negativeselection using B220 magnetic beads (Miltenyi Biotec) followed bypositive selection using CD11-c magnetic beads (Milteny Biotec). Fivethousand CD4⁺ T-cells were then mixed with five thousand dendritic cellsin a 96 well plate in the presences of 1 μM OVA peptide using RPMI (55μM BME, 10% FBS plus antibiotics) in a final 200 uL volume. These cellswere then incubated for 48 to 72 hours in a CO₂ incubator at 37° C. andpulsed using [³H] thymidine for 8 hours. Cells were then harvested and[³H] thymidine incorporation was quantified using a top counter.

Results:

In some assays, anti-CD83 antibodies decreased production of IL-4 byactivated CD4⁺ T-cells in a dose dependent manner. Different antibodypreparations did provide somewhat different degrees of inhibition ofIL-4 production (FIG. 11). Accordingly, the epitope and/or degree ofaffinity of the antibodies for the CD83 antigen may influence whether ornot IL-4 production is significantly inhibited.

The effects of anti CD83 antibodies on proliferation of a peptidespecific T-cell proliferation assay using the OT2 T-cell receptor (TCR)transgenic system were also observed. CD4⁺ T-cells derived from theseTCR transgenic animals express high levels of a T-cell receptor specificfor chicken ovalbumin (OVA) 323-339 peptide and thus have high levels ofproliferation when mixed with antigen presenting cells (dendritic cellswere used) in the presence of the OVA peptide. In such assays, anti-CD83antibodies were able to decrease proliferation of CD4⁺ T-cells in thissystem (FIG. 12). However, different antibody preparations had somewhatdifferent effects on the proliferation of CD4⁺ T-cells. Accordingly, theCD83 epitope and/or degree of affinity of the antibodies for the CD83antigen may influence whether or not CD4⁺ T-cell proliferation issignificantly inhibited.

Example 5 Increased T-Cell Proliferation By Transgenic Expression ofCD83

This Example illustrates that over expression of CD83 in transgenic miceleads to increased T-cell proliferation.

Materials and Methods

A 34.3 kb fragment of normal mouse genomic DNA, including the ˜18 kbcoding region of the CD83 gene, as well as ˜10.6 kb of upstream flankingsequences and ˜5.7 kb of downstream sequences was microinjected intonormal mouse one-cell embryos. Four individual founder animals weregenerated. Transgenic mice were then crossed to a male OT2tg mouse. Maleoffspring carrying both the CD83 and OT2 transgene were used to analyzepeptide specific T-cell proliferation.

For proliferation assays, CD4⁺ T-cells and dendritic cells were isolatedfrom either OT2tg [transgenic mice with a T-cell receptor specific forchicken ovalbumin (OVA) 323-339 peptide] CD83 wild type or from OT2tgCD83 transgenic mice as described above (Example 4). Five thousand OT2tgCD4⁺ T-cells from either wild type or CD83 transgenic animals were thenmixed with five thousand wild type dendritic cells or five thousand CD83transgenic dendritic cells in a 96 well plate in the presence ofincreasing concentrations of OVA peptide using RPMI (55 μM BME, 10% FBSplus antibiotics) in a final 200 uL volume. These cells were thenincubated for 48 to 72 hours in a CO₂ incubator at 37 C and pulsed using[³H] thymidine for 8 hours. Cells were then harvested and [³H] thymidineincorporation was quantified using a top counter.

Results:

OT2tg CD4⁺ T-cells derived from CD83 transgenic mice proliferated athigher rates than the same cell population derived from a CD83 wild typeanimal (FIG. 13). This increased proliferation was seen at all theconcentrations of OVA peptide tested. Whereas OT2tg CD4⁺ T-cells derivedfrom CD83 transgenic animals exhibited increased proliferation,dendritic cells from CD83 transgenic animals did not exhibit asubstantial increase in proliferation. Therefore, it appears thattransgenic expression in the CD4⁺ T-cell, and not in dendritic cells iswhat led to the increased proliferation of CD4⁺ T-cells.

Example 6 Inhibition of Proliferation of PHA Activated Human PBMCs ByProtein A Purified Rabbit Anti Mouse CD83 Polyclonal Sera

This Example shows that antibodies raised against the mouse CD83 proteincan inhibit proliferation of human peripheral blood mononuclear cells.

Materials and Methods

Rabbit polyclonal sera was raised against mouse CD83 protein byimmunizing rabbits using a mouse CD83 external domain protein fused to arabbit Ig domain (FIG. 14). Pre-immune sera and anti-mouse polyclonalsera were then purified using a protein A column (Pharmacia Biotech) asdescribed by the manufacturer, then dialyzed against PBS and stored at4° C. To monitor the recognition of mouse CD83 protein by the polyclonalsera, which was obtained at different dates post immunization, a titerwas obtained using an antigen specific ELISA (FIG. 15). As illustratedby FIG. 15, a good polyclonal response was obtained against the mouseCD83 protein.

Human peripheral blood mononuclear cells (PBMCs) were isolated using aFicoll gradient (Ficoll Paque Plus, Pharmacia) and washed with PBSbuffer. For activation and proliferation studies, five thousand cellswere incubated in 200 μL of media (RPMI, 10% FBS, antibiotics) and 5ug/mL of Phaseolus vulgaris leucoagglutinin (PHA) in the presence orabsence of increasing concentrations of Protein A purified pre-immunesera or with similarly purified anti-CD83 polyclonal antibodies. After48 hours at 37° C. in a CO₂ incubator the cells were pulsed with [³H]thymidine for ˜8 hours and harvested. Thymidine incorporation into thePBMCs was measured using a top counter for analysis.

Results

FIG. 16 illustrates that proliferation of PHA-activated human PBMCs wasinhibited by antibodies raised against the external region of the mouseCD83 protein. Proliferation of PHA-activated human PBMCs was notaffected by addition of increasing concentrations of protein A purifiedrabbit pre-immune sera. When increasing concentrations of protein Apurified rabbit polyclonal sera raised against the mouse CD83 proteinwas added, a concentration dependent decrease in proliferation wasobserved.

These data indicate that antibodies raised against the mouse protein areable to cross-react with the human protein. Moreover, antibodies raisedagainst the mouse protein are able to inhibit proliferation ofPHA-activated human PBMCs.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1-62. (canceled)
 63. An antibody that can bind to a CD83 polypeptide ofSEQ ID NO:9, wherein activated CD4⁺ T-cells produce lower levels ofinterleukin-4 when said T-cells are contacted with the antibody, whereinsaid antibody comprises a light chain and a heavy chain, and wherein theantibody comprises a light chain sequence set forth in SEQ ID NO:15. 64.An antibody that can bind to a CD83 polypeptide of SEQ ID NO:9, whereinCD4⁺ T-cell proliferation is decreased when said T-cells are contactedwith the antibody, wherein said antibody comprises a light chain and aheavy chain, and wherein the antibody comprises a heavy chain sequenceset forth in SEQ ID NO:17.
 65. The antibody of claim 64, wherein theheavy chain variable region CDR1 of the antibody comprises the sequenceDYDLS (SEQ ID NO:25).
 66. An antibody that can bind to a CD83polypeptide of SEQ ID NO:9, wherein activated CD4⁺ T-cells produce lowerlevels of interleukin-4 when said T-cells are contacted with theantibody, wherein said antibody comprises a light chain and a heavychain, and wherein the antibody comprises the heavy chain sequence setforth in SEQ ID NO:17.
 67. An antibody that can bind to a CD83polypeptide of SEQ ID NO:9, wherein CD4⁺ T-cell proliferation isdecreased when said T-cells are contacted with the antibody, whereinsaid antibody comprises a light chain and a heavy chain, and wherein theantibody comprises the heavy chain sequence set forth in SEQ ID NO:17.68. An antibody that modulates the activity or expression of a CD83polypeptide of SEQ ID NO:9, wherein said antibody comprises a lightchain and a heavy chain, and wherein the antibody comprises the heavychain sequence set forth in SEQ ID NO:17.
 69. An antibody according toclaim 67 comprising a light chain variable region comprising a CDR1sequence of RCADVV (SEQ ID NO:38).
 70. An antibody according to claim67, wherein the light chain comprises the sequence of SEQ ID NO:15. 71.An antibody that modulates the activity or expression of a CD83polypeptide of SEQ ID NO:9, wherein said antibody comprises a lightchain and a heavy chain, and wherein the antibody comprises the lightchain sequence set forth in SEQ ID NO:15.
 72. The antibody of claim 71,wherein the heavy chain variable region CDR1 of the antibody comprisesthe sequence RCADVV (SEQ ID NO:38).
 73. The antibody of claim 71,wherein the heavy chain variable region CDR2 of the antibody comprisesthe sequence KNVYNNNW (SEQ ID NO:43).
 74. The antibody of claim 71,wherein the heavy chain variable region CDR3 of the antibody comprisesthe sequence AGDYSSSSDNG (SEQ ID NO:47).
 75. The antibody of claim 71,wherein the heavy chain variable region comprises one or more of theCDR1 of the antibody comprising sequence RCADVV (SEQ ID NO:38), the CDR2of the antibody comprising sequence KNVYNNNW (SEQ ID NO:43), or the CDR3of the antibody comprising sequence AGDYSSSSDNG (SEQ ID NO:47).
 76. Theantibody of claim 63, wherein the heavy chain variable region CDR2 ofthe antibody comprises the sequence AIDGNPYY (SEQ ID NO:30).
 77. Theantibody of claim 63, wherein the heavy chain variable region CDR3 ofthe antibody comprises the sequence GAGD (SEQ ID NO:35).
 78. Theantibody of claim 63, wherein the heavy chain variable region comprisesone or more of the CDR1 of the antibody comprising sequence DYDLS (SEQID NO:25), the CDR2 of the antibody comprising sequence AIDGNPYY (SEQ IDNO:30) or the CDR3 of the antibody comprising sequence GAGD (SEQ IDNO:35).
 79. The antibody of claim 63, wherein the heavy chain variableregion CDR1 of the antibody comprises the sequence DYDLS (SEQ ID NO:25).80. The antibody of claim 64, wherein the heavy chain variable regionCDR2 of the antibody comprises the sequence AIDGNPYY (SEQ ID NO:30). 81.The antibody of claim 64, wherein the heavy chain variable region CDR3of the antibody comprises the sequence GAGD (SEQ ID NO:35).
 82. Theantibody of claim 64, wherein the heavy chain variable region comprisesone or more of the CDR1 of the antibody comprising sequence DYDLS (SEQID NO:25), the CDR2 of the antibody comprising sequence AIDGNPYY (SEQ IDNO:30) or the CDR3 of the antibody comprising sequence GAGD (SEQ IDNO:35).
 83. The antibody of claim 66 wherein the light chain variableregion CDR1 of the antibody comprises the sequence RCADVV (SEQ IDNO:38).
 84. The antibody of claim 66 wherein the light chain variableregion CDR2 of the antibody comprises the sequence KNVYNNNW (SEQ IDNO:43).
 85. The antibody of claim 66 wherein the light chain variableregion CDR3 of the antibody comprises the sequence AGDYSSSSDNG (SEQ IDNO:47).
 86. The antibody of claim 66, wherein the light chain variableregion comprises one or more of the CDR1 of the antibody comprisingsequence RCADVV (SEQ ID NO:38), the CDR2 of the antibody comprisingsequence KNVYNNNW (SEQ ID NO:43), or the CDR3 of the antibody comprisingsequence AGDYSSSSDNG (SEQ ID NO:47).
 87. The antibody of claim 66,wherein the light chain comprises the sequence set forth in SEQ IDNO:15.
 88. The antibody of claim 67 wherein the light chain variableregion CDR1 of the antibody comprises the sequence RCADVV (SEQ IDNO:38).
 89. The antibody of claim 67 wherein the light chain variableregion CDR2 of the antibody comprises the sequence KNVYNNNW (SEQ IDNO:43).
 90. The antibody of claim 67 wherein the light chain variableregion CDR3 of the antibody comprises the sequence AGDYSSSSDNG (SEQ IDNO:47).
 91. The antibody of claim 67, wherein the light chain variableregion comprises one or more of the CDR1 of the antibody comprisingsequence RCADVV (SEQ ID NO:38); the CDR2 of the antibody comprisingsequence KNVYNN (SEQ ID NO:43), or the CDR3 of the antibody comprisingsequence AGDYSSSSDNG (SEQ ID NO:47).
 92. The antibody of claim 67,wherein the light chain comprises the sequence set forth in SEQ IDNO:15.
 93. An antibody according to claim 68 comprising a light chainvariable region comprising a CDR2 sequence of KNVYYNNNW (SEQ ID NO:43).94. An antibody according to claim 68 comprising a light chain variableregion comprising a CDR3 sequence of AGDYSSSSDNG (SEQ ID NO:47).
 95. Theantibody of claim 68, wherein the light chain variable region comprisesone or more of the CDR1 of the antibody comprising sequence RCADVV (SEQID NO:38); the CDR2 of the antibody comprising sequence KNVYYNNNW (SEQID NO:43); and the CDR3 of the antibody comprising sequence AGDYSSSSDNG(SEQ ID NO:47).